Combinations of phosphoinositide 3-kinase inhibitor compounds and chemotherapeutic agents for the treatment of hematopoietic malignancies

ABSTRACT

Combinations of PI3K inhibitor compounds having Formula I and chemotherapeutic agents, including stereoisomers, geometric isomers, tautomers, metabolites and pharmaceutically acceptable salts thereof, are useful for treating hematopoietic malignancies. Methods of using such combinations for in vitro, in situ, and in vivo diagnosis, prevention or treatment of such disorders in mammalian cells, or associated pathological conditions, are disclosed

CROSS REFERENCE TO RELATED APPLICATIONS

This non-provisional application filed under 37 CFR §1.53(b) is adivisional of U.S. Ser. No. 12/721,645, filed on 11 Mar. 2010 and alsoclaims the benefit under 35 USC §119(e) of U.S. Provisional ApplicationSer. No. 61/159,622 filed on 12 Mar. 2009, which is incorporated byreference in entirety.

FIELD OF THE INVENTION

The invention relates generally to pharmaceutical combinations ofcompounds with activity against hematopoietic malignancies and whichinclude compounds that inhibit PI3 kinase or mTOR activity. Theinvention also relates to methods of using the compounds for in vitro,in situ, and in vivo diagnosis or treatment of mammals and mammaliancells.

BACKGROUND OF THE INVENTION

Cancers which involve cells generated during hematopoiesis, a process bywhich cellular elements of blood, such as leukocytes, lymphocytes,natural killer cells, plasma cells, and myeloid cells such asneutrophils and monocytes are generated, are referred to ashematopoietic malignancies. Lymphocytes which can be found in blood andlymphatic tissue and are critical for immune response are categorizedinto two main classes of lymphocytes: B lymphocytes (B cells) and Tlymphocytes (T cells), which mediate humoral and cellular immunity,respectively. B cells are lymphocytes that play a large role in thehumoral immune response (as opposed to the cell-mediated immuneresponse, which is governed by T cells). The principal functions of Bcells are to make antibodies against antigens, perform the role ofAntigen Presenting Cells (APCs) and eventually develop into memory Bcells after activation by antigen interaction. B cells are an essentialcomponent of the adaptive immune system. B cells mature within the bonemarrow and leave the marrow expressing an antigen-binding antibody ontheir cell surface. When a naive B cell first encounters the antigen forwhich its membrane-bound antibody is specific, the cell begins to dividerapidly and its progeny differentiate into memory B cells and effectorcells called “plasma cells”. Memory B cells have a longer life span andcontinue to express membrane-bound antibody with the same specificity asthe original parent cell. Plasma cells do not produce membrane-boundantibody but instead produce the antibody in a form that can besecreted. Secreted antibodies are the major effector molecules ofhumoral immunity.

The non-Hodgkin lymphomas are a diverse group of hematopoieticmalignancies which encompass any lymphoma other than Hodgkin lymphoma.Lymphoma is a type of cancer derived from lymphocytes, a type of whiteblood cell. Many subtypes of non-Hodgkin lymphoma have been described;these are generally grouped by their aggressiveness. Less aggressivenon-Hodgkin lymphomas may be chronic diseases which exist for manyyears, while more aggressive non-Hodgkin lymphomas can be rapidly fatalwithout treatment. Non-Hodgkin lymphomas are treated by combinations ofchemotherapy, monoclonal antibodies, immunotherapy, radiation, andhematopoietic stem cell transplantation.

Lymphoma is a type of neoplasm that originates in lymphocytes (a type ofwhite blood cell in the vertebrate immune system) and in lymph nodes,presenting as an enlargement of the node (a tumor). Lymphomas areclosely related to lymphoid leukemias, which also originate inlymphocytes but do not form solid tumors. There are many types oflymphomas, and in turn, lymphomas are a part of the broad group ofhematopoietic malignancies called hematological neoplasms.

Acute Myeloid Leukemia (AML) comprises a heterogeneous group ofmalignant clonal disorders of hematopoietic stem cells committed to themyeloid linage development. There is a block in normal hematopoieticdifferentiation, often combined with deregulation of proliferation andapoptosis. This leads to progressive insufficiency of the normalhematopoiesis ensuing in anemia, neutropenia and thrombocytopenia. AMLaccounts for about 80% of all adult leukemia, and its overall incidencehas been stable or slowly increasing over the past 15-20 years. Theprognosis of AML remains poor, with an overall 5-year survival rate of15-30%, while patients with AML arising out of myelodysplastic syndromeor who are aged above 60 years have an even worse prognosis with lessthan 10% survival at 5 years (Smith M. et al (2004) Crit. Rev. Oncol.Hematol. 50:197-222). The standard therapeutic approach for AML patientsis high-dose chemotherapy, mainly consisting of cytarabine (Ara-C) andan anthracycline antibiotic such as daunorubicin or idarubicin. Usually,AML responds to initial chemotherapy, but disease relapse occurs in mostpatients. While results of AML treatment have improved in youngerpatients who can tolerate intensified treatment strategies, there havebeen limited changes in outcome among individuals who are above 60 yearsof age. The limit of acceptable toxicity for standard chemotherapeuticdrugs used in AML has been reached. A significant unmet need thereforeremains for new, rationally designed, minimally toxic, and effectivetherapies for AML (Fathi A. T. and Karp J. E. (2009) Curr. Oncol. Rep.11:346-352; Stapnes et al (2009) Expert Opin. Investig. Drugs18:433-455).

Combinations of anti-cancer pharmaceutical therapeutics administeredsimultaneously or sequentially in a dosing regimen are now common incancer treatment. Successful combination therapy provides improved andeven synergistic effect over monotherapy, i.e. pharmaceutical treatmentlimited to one drug (Ouchi et al (2006) Cancer Chemother. Pharmacol.57:693-702; Higgins et al (2004) Anti-Cancer Drugs 15:503-512).Preclinical research has been the basis for prediction of clinical stagesynergy of anti-cancer pharmaceutical therapeutic combinations such ascapecitabine and taxanes for the treatment of breast cancer (Sawada etal (1998) Clin. Cancer Res. 4:1013-1019). Certain doses and schedules ofcombination therapy can improve safety without compromising efficacy(O'Shaughnessy et al (2006) Clin. Breast Cancer April 7(1):42-50).Synergistic effects in vitro have been correlated with clinical stagesynergy (Steinbach et al (2003) Clin. Inf. Dis. October 1:37 Suppl3:S188-224).

Phosphatidylinositol 3-Kinase (PI3K) is a major signaling node for keysurvival and growth signals for lymphomas and is opposed by the activityof the phosphatase PTEN. The PI3K pathway is dysregulated in aggressiveforms of lymphoma (Abubaker (2007) Leukemia 21:2368-2370). Eight percentof DLBCL (diffuse large B-cell lymphoma) cancers have PI3CA(phosphatidylinositol-3 kinase catalytic subunit alpha) missensemutations and 37% are PTEN negative by immunohistochemistry test.

Phosphatidylinositol is one of a number of phospholipids found in cellmembranes, and which participate in intracellular signal transduction.Cell signaling via 3′-phosphorylated phosphoinositides has beenimplicated in a variety of cellular processes, e.g., malignanttransformation, growth factor signaling, inflammation, and immunity(Rameh et al (1999) J. Biol Chem. 274:8347-8350). The enzyme responsiblefor generating these phosphorylated signaling products,phosphatidylinositol 3-kinase (also referred to as PI 3-kinase or PI3K),was originally identified as an activity associated with viraloncoproteins and growth factor receptor tyrosine kinases thatphosphorylate phosphatidylinositol (PI) and its phosphorylatedderivatives at the 3′-hydroxyl of the inositol ring (Panayotou et al(1992) Trends Cell Biol 2:358-60). Phosphoinositide 3-kinases (PI3K) arelipid kinases that phosphorylate lipids at the 3-hydroxyl residue of aninositol ring (Whitman et al (1988) Nature, 332:664). The3-phosphorylated phospholipids (PIP3s) generated by PI3-kinases act assecond messengers recruiting kinases with lipid binding domains(including plekstrin homology (PH) regions), such as Akt and PDK1,phosphoinositide-dependent kinase-1 (Vivanco et al (2002) Nature Rev.Cancer 2:489; Phillips et al (1998) Cancer 83:41).

The PI3 kinase family comprises at least 15 different enzymessub-classified by structural homology and are divided into 3 classesbased on sequence homology and the product formed by enzyme catalysis.The class I PI3 kinases are composed of 2 subunits: a 110 kd catalyticsubunit and an 85 kd regulatory subunit. The regulatory subunits containSH2 domains and bind to tyrosine residues phosphorylated by growthfactor receptors with a tyrosine kinase activity or oncogene products,thereby inducing the PI3K activity of the p110 catalytic subunit whichphosphorylates its lipid substrate. Class I PI3 kinases are involved inimportant signal transduction events downstream of cytokines, integrins,growth factors and immunoreceptors, which suggests that control of thispathway may lead to important therapeutic effects such as modulatingcell proliferation and carcinogenesis. Class I PI3Ks can phosphorylatephosphatidylinositol (PI), phosphatidylinositol-4-phosphate, andphosphatidylinositol-4,5-biphosphate (PIP2) to producephosphatidylinositol-3-phosphate (PIP),phosphatidylinositol-3,4-biphosphate, andphosphatidylinositol-3,4,5-triphosphate, respectively. Class II PI3Ksphosphorylate PI and phosphatidylinositol-4-phosphate. Class III PI3Kscan only phosphorylate PI. A key PI3-kinase isoform in cancer is theClass I PI3-kinase, p110α as indicated by recurrent oncogenic mutationsin p110α (Samuels et al (2004) Science 304:554). (U.S. Pat. No.5,824,492; U.S. Pat. No. 5,846,824; U.S. Pat. No. 6,274,327). Otherisoforms may be important in cancer and are also implicated incardiovascular and immune-inflammatory disease (Workman P (2004) BiochemSoc Trans 32:393-396; Patel et al (2004) Proc. Am. Assoc. of Cancer Res.(Abstract LB-247) 95th Annual Meeting, March 27-31, Orlando, Fla., USA;Ahmadi K and Waterfield Md. (2004) “Phosphoinositide 3-Kinase: Functionand Mechanisms” Encyclopedia of Biological Chemistry (Lennarz W J, LaneM D eds) Elsevier/Academic Press), Oncogenic mutations of p110 alphahave been found at a significant frequency in colon, breast, brain,liver, ovarian, gastric, lung, and head and neck solid tumors. PTENabnormalities are found in glioblastoma, melanoma, prostate,endometrial, ovarian, breast, lung, head and neck, hepatocellular, andthyroid cancers.

PI3 kinase is a heterodimer consisting of p85 and p110 subunits (Otsu etal (1991) Cell 65:91-104; Hiles et al (1992) Cell 70:419-29). Fourdistinct Class I PI3Ks have been identified, designated PI13K α (alpha),β (beta), δ (delta), and ω (gamma), each consisting of a distinct 110kDa catalytic subunit and a regulatory subunit. Three of the catalyticsubunits, i.e., p110 alpha, p110 beta and p110 delta, each interact withthe same regulatory subunit, p85; whereas p110 gamma interacts with adistinct regulatory subunit, p101. The patterns of expression of each ofthese PI3Ks in human cells and tissues are distinct. In each of the PI3Kalpha, beta, and delta subtypes, the p85 subunit acts to localize PI3kinase to the plasma membrane by the interaction of its SH2 domain withphosphorylated tyrosine residues (present in an appropriate sequencecontext) in target proteins (Rameh et al (1995) Cell, 83:821-30; Voliniaet al (1992) Oncogene, 7:789-93).

The PI3 kinase/Akt/PTEN pathway is an attractive target for cancer drugdevelopment since such agents would be expected to inhibit cellularproliferation, to repress signals from stromal cells that provide forsurvival and chemoresistance of cancer cells, to reverse the repressionof apoptosis and surmount intrinsic resistance of cancer cells tocytotoxic agents. PI3 kinase inhibitors have been reported (Yaguchi etal (2006) Jour. of the Nat. Cancer Inst. 98(8):545-556; U.S. Pat. No.7,173,029; U.S. Pat. No. 7,037,915; U.S. Pat. No. 6,608,056; U.S. Pat.No. 6,608,053; U.S. Pat. No. 6,838,457; U.S. Pat. No. 6,770,641; U.S.Pat. No. 6,653,320; U.S. Pat. No. 6,403,588; WO 2006/046031; WO2006/046035; WO 2006/046040; WO 2007/042806; WO 2007/042810; WO2004/017950; US 2004/092561; WO 2004/007491; WO 2004/006916; WO2003/037886; US 2003/149074; WO 2003/035618; WO 2003/034997; US2003/158212; EP 1417976; US 2004/053946; JP 2001247477; JP 08175990; JP08176070). Wortmannin analogs have PI3 kinase activity in mammals (U.S.Pat. No. 6,703,414; WO 97/15658).

Thienopyrimidine compounds of Formula I have p110 alpha binding, PI3kinase inhibitory activity, and inhibit the growth of cancer cells (US2008/0207611; US 2008/0039459; US 2008/0076768; US 2008/0076758; US2008/0242665; US 2008/0269210.

An exemplary Formula I compound, GDC-0941 (CAS Reg. No. 957054-30-7,Genentech Inc.), is a selective, orally bioavailable inhibitor of PI3Kwith promising pharmacokinetic and pharmaceutical properties (Folkes etal (2008) Jour. of Med. Chem. 51(18):5522-5532; US 2008/0076768; Belvinet al, American Association for Cancer Research Annual Meeting 2008,99th: April 15, Abstract 4004; Folkes et al, American Association forCancer Research Annual Meeting 2008, 99th: April 14, Abstract LB-146;Friedman et al, American Association for Cancer Research Annual Meeting2008, 99th: April 14, Abstract LB-110). The exemplary Formula Icompound, GDC-0941, shows synergistic activity in vitro and in vivo incombination with certain chemotherapeutic agents against solid tumorcell lines (U.S. Ser. No. 12/208,227, Belvin et al “Combinations OfPhosphoinositide 3-Kinase Inhibitor Compounds And ChemotherapeuticAgents, And Methods Of Use”, filed 10 Sep. 2008).

SUMMARY OF THE INVENTION

The invention relates generally to thienopyrimidine compounds of FormulaI with anti-cancer activity, and more specifically with PI3 kinase ormTOR inhibitory activity, administered in combination with monoclonalantibody agents or chemotherapeutic agents to inhibit the growth ofhematopoietic malignancies. Certain combinations of Formula I compoundswith chemotherapeutic agents show synergistic effects in inhibiting thegrowth of hematopoietic cancer cells in vitro and in vivo. Thecombinations and methods of the invention may be useful in the treatmentof hematopoietic malignancies. The compositions may inhibit tumor growthin mammals and may be useful for treating human cancer patients.

In one aspect, the invention includes a method for the treatment of ahematopoietic malignancy comprising administering a therapeuticcombination as a combined formulation or alternation to a mammal,wherein the therapeutic combination comprises a therapeuticallyeffective amount of a compound having Formula I, and a therapeuticallyeffective amount of a chemotherapeutic agent selected fromdexamethasone, thioTEPA, doxorubicin, vincristine, rituximab,cyclophosphamide, prednisone, melphalan, lenalidomide, bortezomib,rapamycin, and cytarabine.

The invention also relates to methods of using the therapeuticcombinations for in vitro, in situ, and in vivo diagnosis or treatmentof mammalian cells, organisms, or associated pathological conditionsrelated to the hematopoietic malignancies.

An aspect of the invention provides therapeutic combinations comprising4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazzin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine(US 2008/0076768; US 2008/0207611; Folkes et al (2008) Jour. of Med.Chem. 51(18):5522-5532), also known as GDC-0941 (Genentech, Inc.) andhaving Formula Ia and a therapeutically effective amount of achemotherapeutic agent selected from dexamethasone, thioTEPA,doxorubicin, vincristine, rituximab, cyclophosphamide, prednisone,melphalan, lenalidomide, bortezomib, rapamycin, and cytarabine.

An aspect of the invention provides therapeutic combinations comprising(S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one(US 2008/0242665) having Formula Ib and a therapeutically effectiveamount of a chemotherapeutic agent selected from dexamethasone,thioTEPA, doxorubicin, vincristine, rituximab, cyclophosphamide,prednisone, melphalan, lenalidomide, bortezomib, rapamycin, andcytarabine.

Formula I compounds include all stereoisomers, geometric isomers,tautomers, metabolites, and pharmaceutically acceptable salts thereof.Certain Formula I compounds are potent inhibitors of PI3K with drug-likephysicochemical and pharmacokinetic properties. Certain Formula Icompounds exhibit selectivity for class Ia PI3Ks over class Ib, inparticular for the P110 alpha subtype. Formula Ia and Ib compounds areorally bioavailable and have single agent anti-tumor activity inmultiple human cancer models.

Pharmaceutical compositions and therapeutic combinations of theinvention comprise a chemotherapeutic agent selected from dexamethasone,thioTEPA, doxorubicin, vincristine, rituximab, cyclophosphamide,prednisone, melphalan, lenalidomide, bortezomib, rapamycin, andcytarabine.

Pharmaceutical compositions of the invention may further comprise apharmaceutically acceptable carrier.

Another aspect of the invention provides methods of treating ahematopoietic malignancy modulated by PI3 kinases, comprisingadministering to a mammal in need of such treatment effective amounts ofa Formula I compound and a chemotherapeutic agent. The Formula Icompound and the chemotherapeutic agent may be co-formulated foradministration in a combination as a pharmaceutical composition or theymay be administered separately in alternation (sequentially,consecutively) as a therapeutic combination.

Another aspect of the invention provides methods of treating ahematopoietic malignancy, comprising administering to a mammal in needof such treatment effective amounts of the Formula I compound and achemotherapeutic agent.

In a further aspect the present invention provides a method of using apharmaceutical composition of the invention to treat a hematopoieticmalignancy disease or condition modulated by PI3 kinase in a mammal.

An additional aspect of the invention is the use of a pharmaceuticalcomposition of the invention in the preparation of a medicament for thetreatment of a hematopoietic malignancy disease or condition modulatedby PI3 kinase in a mammal.

Another aspect of the invention includes articles of manufacture or kitscomprising a Formula I compound, a chemotherapeutic agent, a container,and optionally a package insert or label indicating a treatment for ahematopoietic malignancy.

Another aspect of the invention is a product comprising a Formula Icompound, and a chemotherapeutic agent selected from dexamethasone,thioTEPA, doxorubicin, vincristine, rituximab, cyclophosphamide,prednisone, melphalan, lenalidomide, bortezomib, rapamycin, andcytarabine; as a combined preparation for separate, simultaneous orsequential use in the treatment of a hematopoietic malignancy.

Another aspect of the invention includes a method for determiningcompounds to be used in combination for the treatment of cancercomprising: a) administering a therapeutic combination comprising aFormula I compound, and a chemotherapeutic agent to an in vitrohematopoietic malignancy cell line with one or more mutations, and b)measuring a synergistic or non-synergistic effect.

Another aspect of the invention is methods of therapeutically treating amammal having a hematopoietic malignancy, wherein the method comprisesadministering to the mammal a therapeutically effective amount of thetherapeutic combination, thereby resulting in the effective therapeutictreatment of the hematopoietic malignancy, such as non-Hodgkin'slymphoma, diffuse large hematopoietic lymphoma, follicular lymphoma,mantle cell lymphoma, chronic lymphocytic leukemia, multiple myeloma,AML, and MCL. In one embodiment, the therapeutic combination inhibitsone or more isoforms of PI3K. In one embodiment, the therapeuticcombination inhibits mTOR.

In one aspect, the invention provides a method of inhibiting the growthof a non-Hodgkin's lymphoma comprising administering the therapeuticcombination to a patient with a non-Hodgkin's lymphoma, whereby growthof the lymphoma is inhibited.

In one aspect, the invention provides a method of inhibiting the growthof a non-Hodgkin's lymphoma comprising administering the therapeuticcombination to a lymphoma cell, or to a cell present in and/or adjacentto the lymphoma, whereby growth of the lymphoma is inhibited. In oneembodiment, said cell is not a non-Hodgkin's lymphoma cell (e.g., it isnot a T or B cell) for example, said cell may be a stromal cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b shows reduction of pharmacodynamic (PD) markers measuredby flow cytometry with Formula Ia (GDC-0941) treated (right column) anduntreated (left column) cells, DoHH2, WSU-DLCL2, OPM2, and U266. Cellswere treated in vitro with 5 μM GDC-0941 for 4 hrs.

FIG. 2 shows reduction of pharmacodynamic (PD) markers p-AKT, p-S6RP,p-Bad, and beta-actin in cells DoHH2, WSU-DLCL2, OPM2, and U266 asmeasured by SDS-polyacrylamide gel electrophoresis and western blottingin cell lines treated in vitro with 5 μM GDC-0941 for 4 hrs.

FIG. 3 shows the effect of PI3K single agent inhibitor, Formula Ia(GDC-0941), and in combinations with dexamethasone (Dex) and doxorubicin(Dox), on B-NHL cell line DoHH2. In vitro cell survival andproliferation assays (Cell-Titer Glo, Promega) measured viable cellsover varying inhibitor concentrations (10⁻⁵ to 10 Relative Units of thepreviously (approximately) determined IC50, Formula Ia, dexamethasone,doxorubicin and combinations of Formula Ia and dexamethasone; andFormula Ia and doxorubicin.

FIG. 4 shows the effect of PI3K single agent inhibitors on primaryfollicular lymphoma cells from patient NHL600-A876 cells by in vitrocell survival and proliferation assays (Cell-Titer Glo®, Promega Corp.,Madison, Wis.) measuring viable cells over varying concentrations (10-to 10 μMolar) of Formula Ia (GDC-0941), GDC-0464, and LY294002.

FIG. 5 shows the effect of a PI3K single agent inhibitor, Formula Ia(GDC-0941), and in combination with doxorubicin, on primary diffuselarge B-cell lymphoma (DLBCL) cells from patient NHL640-A055. Cellviability was measured by in vitro cell survival and proliferationassays (Cell-Titer Glo®) over varying concentrations (10⁻⁵ to 20 μMolar)of Formula Ia, doxorubicin, and the combination of Formula Ia anddoxorubicin.

FIG. 6 shows the effect of PI3K single agent inhibitor Formula Ia(GDC-0941), and in combination with dexamethasone, on multiple myelomaOPM2 cells by in vitro cell survival and proliferation assays(Cell-Titer Glo®) measuring viable cells over varying concentrations(10⁻⁵ to 10 μMolar) of Formula Ia, dexamethasone, and the combination ofFormula Ia and dexamethasone.

FIG. 7 shows the mean tumor volume change over 20 days in cohorts of 10mice with WSU-DLCL2 lymphoma tumor xenografts dosed on day 0 withVehicle (0.5% Methylcellulose: 0.2% Tween 80 in DI Water), 73 mg/kgFormula Ia (GDC-0941), 5 mg/kg rituximab, CHOP, and the combinations ofFormula Ia 73 mg/kg and rituximab 5 mg/kg, Formula Ia 73 mg/kg and CHOP.Mice were dosed once with CHOP starting on day 0, and rituximab on days0, 7, and 14, while Formula Ia was dosed daily for 21 days by oralgavage. CHOP regimen: cyclophosphamide (30 mg/kg, iv, qd×1), doxorubicin(2.475 mg/kg, iv, qd×1), vincristine (0.375 mg/kg, iv, qd×1), prednisone(0.15 mg/kg, po, qd×5). Cyclophosphamide, doxorubicin and vincristinewere dosed once on day 0 and prednisone was dosed on days 0, 1, 2, 3 and4.

FIG. 8 shows the mean tumor volume change over 35 days in cohorts of 10mice with DoHH-2 lymphoma tumor xenografts dosed on day 0 with: Vehicle(0.5% methylcellulose: 0.2% Tween 80 in DI Water), 73 mg/kg Formula Ia(GDC-0941), 5 mg/kg rituximab, CHOP, and the combinations of Formula Ia73 mg/kg and rituximab 5 mg/kg, and Formula Ia 73 mg/kg and CHOP. Micewere dosed with rituximab on day 0, 7 and 14 (qwk×3) intravenously, CHOPstarting on day 1, while Formula Ia was dosed daily for 21 days by oralgavage. CHOP regimen: cyclophosphamide (30 mg/kg, iv, qd×1), doxorubicin(2.475 mg/kg, iv, qd×1), vincristine (0.375 mg/kg, iv, qd×1), prednisone(0.15 mg/kg, po, qd×5). Cyclophosphamide, doxorubicin and vincristinewere dosed once on day 0 and prednisone was dosed on days 0, 1, 2, 3 and4.

FIG. 9 shows the mean tumor volume change over 27 days in cohorts of 10mice with DoHH2 lymphoma tumor xenografts dosed on day 0 with: Vehicle(0.5% Methylcellulose: 0.2% Tween 80 in DI Water), 75 mg/kg Formula Ia(GDC-0941), CHOP, and the combinations of Formula Ia 75 mg/kg and CHOP,Formula Ia 75 mg/kg and cyclophosphamide 30 mg/kg, Formula Ia 75 mg/kgand doxorubicin 2.47 mg/kg, Formula Ia 75 mg/kg and vincristine 0.38mg/kg, and Formula Ia 75 mg/kg and prednisone 0.15 mg/kg. Mice weredosed with CHOP on day 0, cyclophosphamide on day 0, doxorubicin on day0, vincristine on day 0, and prednisone daily on days 0-4, while FormulaIa was dosed daily for 21 days by oral gavage. CHOP components regimenwas: cyclophosphamide (30 mg/kg, iv, qd×1), doxorubicin (2.475 mg/kg,iv, qd×1), vincristine (0.375 mg/kg, iv, qd×1), prednisone (0.15 mg/kg,po, qd×5)

FIG. 10 shows the mean tumor volume change over 25 days in cohorts of 10mice with BJAB lymphoma tumor xenografts dosed on day 0 with: Vehicle(0.5% Methylcellulose: 0.2% Tween 80 in DI Water), 73 mg/kg Formula Ia(GDC-0941), CHOP, and the combination of Formula Ia 73 mg/kg and CHOP.Mice were dosed once with CHOP starting on day 0, while Formula Ia andVehicle were dosed daily for 21 days by oral gavage. CHOP regimen:cyclophosphamide (30 mg/kg, iv, qd×1), doxorubicin (2.475 mg/kg, iv,qd×1), vincristine (0.375 mg/kg, iv, qd×1), prednisone (0.15 mg/kg, po,qd×5). Cyclophosphamide, doxorubicin and vincristine were dosed once onday 0 and prednisone was dosed on days 0, 1, 2, 3 and 4.

FIG. 11 shows the mean tumor volume change over 25 days in cohorts of 10mice with BJAB lymphoma tumor xenografts dosed on day 0 with: Vehicle(0.5% Methylcellulose: 0.2% Tween 80 in DI Water), 75 mg/kg Formula Ia(GDC-0941), 5 mg/kg rituximab, and the combination of Formula Ia 75mg/kg and 5 mg/kg rituximab. Mice were dosed with rituximab on days 0,7, and 14, while Formula Ia and Vehicle were dosed daily for 21 days(po, qd×21) by oral gavage.

FIG. 12 shows the mean tumor volume change over 22 days in cohorts of 10mice with NCI-H929 multiple myeloma xenografts dosed on day 0 with:Vehicle (po, qd×21) (0.5% Methylcellulose: 0.2% Tween 80 in DI Water),and single agent therapies: 73 mg/kg Formula Ia GDC-0941, 1 mg/kgbortezomib, 25 mg/kg lenalidomide, and 10 mg/kg dexamethasone. FormulaIa GDC-0941 was dosed daily for 21 days by oral gavage. Bortezomib wasdosed intravenously on days 0, 3, 7, 10, 14 and 17. Lenalidomide wasdosed daily for 21 days by intraperitoneal injection. Dexamethasone wasdosed orally on days 0, 1, 2, 3, 4, 7, 8, 9 and 10.

FIG. 13 shows the mean tumor volume change over 24 days in cohorts of 10mice with multiple myeloma OPM-2 cell xenografts dosed on day 0 with:Vehicle (po, qd×21) (0.5% Methylcellulose: 0.2% Tween 80 in DI Water),single agent therapies: 73 mg/kg Formula Ia GDC-0941 (po, qd×21); 0.5mg/kg bortezomib (iv, 2×/wk×3); 25 mg/kg lenalidomide (ip, 5 days on/2days off/5 days on/2 days off/5 days on); and 3 mg/kg dexamethasone (po,5 days on/2 days off/5 days on); and combinations of: 73 mg/kg FormulaIa GDC-0941 (po, qd×21) and 0.5 mg/kg bortezomib (iv, 2×/wk×3); 73 mg/kgFormula Ia GDC-0941 (po, qd×21) and 25 mg/kg lenalidomide (ip,5/2/5/2/5); and 73 mg/kg Formula Ia GDC-0941 (po, qd×21) and 3 mg/kgdexamethasone (po, 5/2/5). Formula Ia GDC-0941 was dosed daily for 21days by oral gavage. Bortezomib was dosed intravenously on days 0, 3, 7,10, 14 and 17. Lenalidomide was dosed on days 0-4, 7-11 and 14-18 byintraperitoneal injection. Dexamethasone was dosed orally on days 0-4and 7-11.

FIG. 14 shows the mean tumor volume change over 27 days in cohorts of 10mice with multiple myeloma MM1.s cell xenografts dosed on day 0 with:Vehicle (po, qd×21) (0.5% Methylcellulose: 0.2% Tween 80 in DI Water),73 mg/kg Formula Ia GDC-0941 (po, qd×21), 1 mg/kg bortezomib (iv,2×/wk×2.5), 25 mg/kg lenalidomide (ip, qd×21), and 10 mg/kgdexamethasone (po, 5 days on/2 days off/5 days on). Formula Ia GDC-0941was dosed daily for 21 days by oral gavage. Bortezomib was dosedintravenously on days 0, 3, 7, and 14. Lenalidomide was dosed daily for21 days by intraperitoneal injection. Dexamethasone was dosed orally ondays 0-4 and 7-11.

FIG. 15 shows the mean tumor volume change over 40 days in cohorts of 10mice with multiple myeloma MM1.s cell xenografts dosed on day 0 with:Vehicle (po, qd×21) (0.5% Methylcellulose: 0.2% Tween 80 in DI Water),single agent therapies: 75 mg/kg Formula Ia GDC-0941 (po, qd×21), 0.5mg/kg bortezomib (iv, 2×/wk×3), and 3 mg/kg dexamethasone (po, 5/2/5);and combinations of: 75 mg/kg Formula Ia GDC-0941 (po, qd×21) and 0.5mg/kg bortezomib (iv, 2×/wk×3); and 75 mg/kg Formula Ia GDC-0941 (po,qd×21) and 3 mg/kg dexamethasone (po, 5/2/5). Formula Ia GDC-0941 wasdosed daily for 21 days by oral gavage. Bortezomib was dosedintravenously on days 0, 3, 7, 10, 14 and 17. Dexamethasone was dosedorally on days 0-4 and 7-11.

FIG. 16 shows the mean tumor volume change over 33 days in cohorts of 10mice with multiple myeloma H929 cell xenografts dosed on day 0 with:Vehicle (po, qd×21) (0.5% Methylcellulose: 0.2% Tween 80 in DI Water)single agent therapies: 75 mg/kg Formula Ia GDC-0941 (po, qd×21), 0.5mg/kg bortezomib (iv, 2×/wk×3), 25 mg/kg lenalidomide (ip, 5/2/5/2/5),and 3 mg/kg dexamethasone (po, 5/2/5); and combinations of: 75 mg/kgFormula Ia GDC-0941 (po, qd×21) and 0.5 mg/kg bortezomib (iv, 2×/wk×3);75 mg/kg Formula Ia GDC-0941 (po, qd×21) and 25 mg/kg lenalidomide (ip,5/2/5/2/5); and 75 mg/kg Formula Ia GDC-0941 (po, qd×14) and 3 mg/kgdexamethasone (po, 5/2/5). Formula Ia GDC-0941 was dosed daily for 21days by oral gavage except when combined with dexamethasone where it wasdosed for 14 days. Bortezomib was dosed intravenously on days 0, 3, 7,10, 14 and 17. Lenalidomide was dosed on days 0-4, 7-11 and 14-18 byintraperitoneal injection. Dexamethasone was dosed orally on days 0-4and 7-11.

FIG. 17 shows the mean tumor volume change over 33 days in cohorts of 10mice with DoHH2 lymphoma tumor xenografts dosed on day 0 with: Vehicle(0.5% Methylcellulose: 0.2% Tween 80 in DI Water), 6 mg/kg rapamycin(ip, qwk×3), 75 mg/kg Formula Ia GDC-0941 (po, qd×21), 100 mg/kg FormulaIa GDC-0941 (po, qd×21), 2.5 mg/kg Formula Ib (po, qd×21), 4 mg/kgFormula Ib (po, qd×21); and combinations of: 6 mg/kg rapamycin (ip,qwk×3) and 75 mg/kg Formula Ia GDC-0941 (po, qd×21); and 6 mg/kgrapamycin (ip, qwk×3) and 2.5 mg/kg Formula Ib (po, qd×21). Formula IaGDC-0941 and Formula Ib were each dosed daily for 21 days by oralgavage. Rapamycin was dosed intravenously on days 0, 7, and 14.

FIG. 18 shows the mean tumor volume change over 21 days in cohorts of 10mice with WSU-DLCL2 lymphoma tumor xenografts dosed on day 0 withVehicle (0.5% Methylcellulose: 0.2% Tween 80 in DI Water), 6 mg/kgrapamycin (ip, qwk×3), 60 mg/kg Formula Ia GDC-0941 (po, qd×21), 1 mg/kgFormula Ib (po, qd×21); and combinations of: 6 mg/kg rapamycin (ip,qwk×3) and 60 mg/kg Formula Ia GDC-0941 (po, qd×18); and 6 mg/kgrapamycin (ip, qwk×3) and 1 mg/kg Formula Ib (po, qd×18).

DEFINITIONS

The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and claims are intended tospecify the presence of stated features, integers, components, or steps,but they do not preclude the presence or addition of one or more otherfeatures, integers, components, steps, or groups thereof.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder, such as the growth, development or spread of cancer. Those inneed of treatment include those already with the disorder as well asthose prone to have the disorder or those in whom the disorder is to beprevented. For purposes of this invention, beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadywith the condition or disorder as well as those prone to have thecondition or disorder or those in which the condition or disorder is tobe prevented.

A subject or mammal is successfully “treated” for a hematopoieticmalignancy, such as non-Hodgkin's lymphoma, if after receiving atherapeutic amount of the therapeutic combination according to themethods of the invention, the patient shows one or more of: (i)observable and/or measurable reduction in the number of cancer cells orabsence of the cancer cells; (ii) reduction in the tumor size;inhibition (i.e., slow to some extent and preferably stop) of cancercell infiltration into peripheral organs including the spread of cancerinto soft tissue and bone; (iii) inhibition (i.e., slow to some extentand preferably stop) of tumor metastasis; (iv) inhibition, to someextent, of tumor growth; or (v) relief to some extent, of one or more ofthe symptoms associated with the specific cancer, including reducedmorbidity and mortality and improvement in quality of life. To theextent the therapeutic combination may prevent growth and/or killexisting cancer cells, it may be cytostatic and/or cytotoxic. Reductionof these signs or symptoms may also be felt by the patient. The aboveparameters for assessing successful treatment and improvement in thedisease are readily measurable by routine procedures familiar to aphysician. For cancer therapy, efficacy can be measured, for example, byassessing the time to disease progression (TTP) and/or determining theresponse rate (RR). Metastasis can be determined by staging tests and bybone scan and tests for calcium level and other enzymes to determinespread to the bone. CT scans can also be done to look for spread to thepelvis and lymph nodes in the area. Chest X-rays and measurement ofliver enzyme levels by known methods are used to look for metastasis tothe lungs and liver, respectively.

The term “hematopoietic malignancy” refers to a cancer orhyperproliferative disorder generated during hematopoiesis involvingcells such as leukocytes, lymphocytes, natural killer cells, plasmacells, and myeloid cells such as neutrophils and monocytes.Hematopoietic Malignancies include the diseases listed in Table 1, theWHO classification of Human Hematopoietic Malignancies; Tumors ofHematopoietic and Lymphoid Tissues (Jaffe E. S., Harris N. L., Stein H.,Vardiman J. W. (Eds.) (2001): World Health Organization Classificationof Tumours. Pathology and Genetics of Tumours of Hematopoietic andLymphoid Tissues. IARC Press: Lyon) with the morphology code of theInternational Classification of Diseases (ICD-O). Behavior is coded /3for malignant tumors and /1 for lesions of low or uncertain malignantpotential.

TABLE 1 I. CHRONIC MYELOPROLIFERATIVE DISEASES Chronic myelogenousleukemia - ICD-O 9875/3 Chronic neutrophilic leukemia - ICD-O 9963/3Chronic eosinophilic leukemia/hypereosinophilic syndrome - ICD-O 9964/3Polycythemia vera - ICD-O 9950/3 Chronic idiopathic myelofibrosis -ICD-O 9961/3 Essential thrombocytemia - ICD-O 9962/3 ChronicMyeloproliferative disease, unclassifiable - ICD-O 9975/3 II.MYELODYSPLASTIC/MYELOPROLIFERATIVE DISEASES Chronic myelomonocyticleukemia - ICD-O 9980/3 Atypical chronic myelogenous leukemia - ICD-O9876/3 Juvenile myelomonocytic leukemia - ICD-O 9946/3Myelodysplastic/myeloproliferative diseases, unclassifiable - ICD-O9975/3 III. MYELODYSPLASTIC SYNDROMES Refractory anemia - ICD-O 9980/3Refractory anemia with ringed sideroblasts - ICD-O 9982/3 Refractorycytopenia with multilineage dysplasia - ICD-O 9985/3 Refractory anemiawith excess blasts - ICD-O 9983/3 Myelodysplastic syndrome associatedwith isolated del(5q) chromosome abnormality - ICD-O 9986/3Myelodysplastic syndrome, unclassifiable 9989/3 IV. ACUTE MYELOIDLEUKEMIAS Acute myeloid leukemias with recurrent cytogeneticabnormalities AML with t(8; 21)(q22; q22), AML1/ETO - ICD-O 9896/3 AMLwith inv(16)(p13q22) or t(16; 16)(p13; q22), CBFb/MYH11 - ICD-O 9871/3Acute promyelocytic leukemia (AML with t(15; 17)(q22; q12), PML- RARaand variants) - ICD-O 9866/3 AML with 11q23 (MLL) abnormalities - ICD-O9897/3 Acute myeloid leukemia multilineage dysplasia- ICD-O 9895/3 Acutemyeloid leukemia and myelodysplastic syndrome, therapy related - ICD-O9920/3 Acute myeloid leukemia not otherwise categorised Acute myeloidleukemia, minimally differentiated - ICD-O 9872/3 Acute myeloidleukemia, without maturation - ICD-O 9873/3 Acute myeloid leukemia, withmaturation - ICD-O 9874/3 Acute myelomonocytic leukemia - ICD-O 9867/3Acute monoblastic and monocytic leukemia - ICD-O 9891/3 Acute erythroidleukemia - ICD-O 9840/3 Acute megakaryoblastic leukemia - ICD-O 9910/3Acute basophilic leukemia - ICD-O 9870/3 Acute panmyelosis withmyelofibrosis - ICD-O 9931/3 Myeloid sarcoma - ICD-O 9930/3 Acuteleukemia of ambiguous lineage - ICD-O 9805/3 V. B-CELL NEOPLASMSPrecursor hematopoietic neoplasm Precursor B lymphoblastic leukemia/-ICD-O 9835/3 lymphoma - ICD-O 9728/3 Mature hematopoietic neoplasmChronic lymphocytic leukemia/- ICD-O 9823/3 small lymphocytic lymphoma -ICD-O 9670/3 hematopoietic prolymphocytic leukemia - ICD-O 9833/3Lymphoplasmacytic lymphoma - ICD-O 9671/3 Splenic marginal zonelymphoma - ICD-O 9689/3 Hairy cell leukemia - ICD-O 9940/3 Plasma cellmyeloma - ICD-O 9732/3 Solitary plasmacytoma of bone - ICD-O 9731/3Extraosseous plasmacytoma - ICD-O 9734/3 Extranodal marginal zonehematopoietic lymphoma of mucosa- associated lymphoid tissue(MALT-lymphoma) - ICD-O 9699/3 Nodal marginal zone hematopoieticlymphoma - ICD-O 9699/3 Follicular lymphoma - ICD-O 9690/3 Mantle celllymphoma - ICD-O 9673/3 Diffuse large hematopoietic lymphoma - ICD-O9680/3 Mediastinal (thymic) large cell lymphoma - ICD-O 9679/3Intravascular large hematopoietic lymphoma - ICD-O 9680/3 Primaryeffusion lymphoma - ICD-O 9678/3 Burkitt lymphoma/- ICD-O 9687/3leukemia - ICD-O 9826/3 hematopoietic proliferations of uncertainmalignant potential Lymphomatoid granulomatosis - ICD-O 9766/1Post-transplant lymphoproliferative disorder, pleomorphic - ICD-O 9970/1VI. T-CELL AND NK-CELL NEOPLASMS Precursor T-cell neoplasms Precursor Tlymphoblastic leukemia/- ICD-O 9837/3 lymphoma - ICD-O 9729/3 Blastic NKcell lymphoma - ICD-O 9727/3 Mature T-cell and NK-cell neoplasms T-cellprolymphocytic leukemia - ICD-O 9834/3 T-cell large granular lymphocyticleukemia - ICD-O 9831/3 Aggressive NK cell leukemia - ICD-O 9948/3 AdultT-cell leukemia/lymphoma - ICD-O 9827/3 Extranodal NK/T cell lymphoma,nasal type - ICD-O 9719/3 Enteropathy type T-cell lymphoma - ICD-09717/3 Hepatosplenic T-cell lymphoma - ICD-O 9716/3 Subcutaneouspanniculitis-like T-cell lymphoma - ICD-O 9708/3 Mycosis fungoides -ICD-O 9700/3 Sezary Syndrome - ICD-O 9701/3 Primary cutaneous anaplasticlarge cell lymphoma - ICD-O 9718/3 Peripheral T-cell lymphoma,unspecified -ICD-O 9702/3 Angioimmunoblastic T-cell lymphoma - ICD-O9705/3 Anaplastic large cell lymphoma - ICD-O 9714/3 T-cellproliferation of uncertain malignant potential Lymphomatoid papulosis -ICD-O 9718/1 VII. HODGKIN LYMPHOMA Nodular lymphocyte predominantHodgkin lymphoma - ICD-O 9659/3 Classical Hodgkin lymphoma - ICD-O9650/3 Nodular sclerosis classical Hodgkin lymphoma - ICD-O 9663/3Lymphocyte-rich classical Hodgkin lymphoma - ICD-O 9651/3 Mixedcellularity classical Hodgkin lymphoma - ICD-O 9652/3Lymphocyte-depleted classical Hodgkin lymphoma - ICD-O 9653/3 VIII.HISTIOCYTIC AND DENDRITIC-CELL NEOPLASMS Macrophage/histiocytic neoplasmHistiocytic sarcoma - ICD-O 9755/3 Dendritic cell neoplasms Langerhanscell histiocytosis - ICD-O 9751/1 Langerhans cell sarcoma - ICD-O 9756/3Interdigitating dendritic cell sarcoma/tumor - ICD-O 9757/3/1 Folliculardendritic cell sarcoma/tumor - ICD-O 9758/3/1 Dendritic cell sarcoma,not otherwise specified - ICD-O 9757/3 IX. MASTOCYTOSIS Cutaneousmastocytosis Indolent systemic mastocytosis - ICD-O 9741/1 Systemicmastocytosis with associated clonal, hematological non- mast celllineage disease - ICD-O 9741/3 Aggressive systemic mastocytosis - ICD-O9741/3 Mast cell leukemia - ICD-O 9742/3 Mast cell sarcoma - ICD-O9740/3 Extracutaneous mastocytoma - ICD-O 9740/1

A “B cell” is a lymphocyte that matures within the bone marrow, andincludes a naïve B cell, memory B cell, or effector B cell (plasmacell). The B cell herein is a normal or non-malignant B cell.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,multispecific antibodies (e.g. bispecific antibodies) formed from atleast two intact antibodies, and antibody fragments, so long as theyexhibit the desired biological activity.

The phrase “therapeutically effective amount” means an amount of acompound of the present invention that (i) treats the particulardisease, condition, or disorder, (ii) attenuates, ameliorates, oreliminates one or more symptoms of the particular disease, condition, ordisorder, or (iii) prevents or delays the onset of one or more symptomsof the particular disease, condition, or disorder described herein. Inthe case of cancer, the therapeutically effective amount of the drug mayreduce the number of cancer cells; reduce the tumor size; inhibit (i.e.,slow to some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic, as measured by TTPand/or response rate (RR).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include squamous cell cancer (e.g.,epithelial squamous cell cancer), lung cancer including small-cell lungcancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lungand squamous carcinoma of the lung, cancer of the peritoneum,hepatocellular cancer, gastric or stomach cancer includinggastrointestinal cancer, pancreatic cancer, glioblastoma, cervicalcancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breastcancer, colon cancer, rectal cancer, colorectal cancer, endometrial oruterine carcinoma, salivary gland carcinoma, kidney or renal cancer,prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, analcarcinoma, penile carcinoma, as well as head and neck cancer.

“Chronic” administration refers to administration of the agent(s) in acontinuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

“Mammal” for purposes of the treatment of, alleviating the symptoms of acancer refers to any animal classified as a mammal, including humans,domestic and farm animals, and zoo, sports, or pet animals, such asdogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.Preferably, the mammal is human.

Administration “in combination with” one or more further therapeuticagents includes simultaneous (concurrent) and consecutive (alternation)administration in any order.

A “chemotherapeutic agent” is a biological (large molecule) or chemical(small molecule) compound useful in the treatment of cancer, regardlessof mechanism of action. Classes of chemotherapeutic agents include, butare not limited to: alkylating agents, antimetabolites, spindle poisonplant alkaloids, cytotoxic/antitumor antibiotics, topoisomeraseinhibitors, proteins, antibodies, photosensitizers, and kinaseinhibitors. Chemotherapeutic agents include compounds used in “targetedtherapy” and non-targeted, conventional chemotherapy.

Examples of chemotherapeutic agents include: dexamethasone, thioTEPA,doxorubicin, vincristine, rituximab, cyclophosphamide, prednisone,melphalan, lenalidomide, bortezomib, rapamycin, and cytarabine.

Examples of chemotherapeutic agents also include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin(CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers SquibbOncology), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carboxamide,CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, rapamycin, and lapatinib (TYKERB®, Glaxo SmithKline).

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (MEK inhibitor, Exelixis, WO2007/044515), ARRY-886 (MEK inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), ABT-869(multi-targeted inhibitor of VEGF and PDGF family receptor tyrosinekinases, Abbott Laboratories and Genentech), ABT-263 (Bcl-2/Bcl-xLinhibitor, Abbott Laboratories and Genentech), PTK787/ZK 222584(Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinicacid), lonafamib (SARASAR™, SCH 66336, Schering Plough), sorafenib(NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca),irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifamib (ZARNESTRA™, Johnson& Johnson), capecitabine (XELODA®, Roche), ABRAXANE™ (Cremophor-free),albumin-engineered nanoparticle formulations of paclitaxel (AmericanPharmaceutical Partners, Schaumberg, Ill.), vandetanib (rINN, ZD6474,ZACTIMA®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen),temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline),canfosfamide (TELCYTA®, Telik), thioTepa and cyclosphosphamide(CYTOXAN®, NEOSAR®); alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlomaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, calicheamicin gamma1I, calicheamicin omegaI1, dynemicin,dynemicin A; bisphosphonates, such as clodronate; an esperamicin; aswell as neocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, caminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, porfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thioTepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such asretinoic acid; and pharmaceutically acceptable salts, acids andderivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, rhuMab 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the PI3K inhibitors of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

The term “mammal” includes, but is not limited to, humans, mice, rats,guinea pigs, monkeys, dogs, cats, horses, cows, pigs, and sheep, andpoultry.

The term “alkyl” as used herein refers to a saturated linear orbranched-chain monovalent hydrocarbon radical of one to twelve carbonatoms, wherein the alkyl radical may be optionally substitutedindependently with one or more substituents described below. Examples ofalkyl groups include, but are not limited to, methyl (Me, —CH₃), ethyl(Et, —CH₂CH₃), 1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr,i-propyl, —CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃),2-methyl-1-propyl (1-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl,—CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl(n-pentyl, —CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, 1-heptyl, 1-octyl, and the like.

The term “alkenyl” refers to linear or branched-chain monovalenthydrocarbon radical of two to twelve carbon atoms with at least one siteof unsaturation, i.e., a carbon-carbon, sp² double bond, wherein thealkenyl radical may be optionally substituted independently with one ormore substituents described herein, and includes radicals having “cis”and “trans” orientations, or alternatively, “E” and “Z” orientations.Examples include, but are not limited to, ethylenyl or vinyl (—CH═CH₂),allyl (—CH₂CH═CH₂), and the like.

The term “alkynyl” refers to a linear or branched monovalent hydrocarbonradical of two to twelve carbon atoms with at least one site ofunsaturation, i.e., a carbon-carbon, sp triple bond, wherein the alkynylradical may be optionally substituted independently with one or moresubstituents described herein. Examples include, but are not limited to,ethynyl (—C≡CH), propynyl (propargyl, —CH₂C≡CH), and the like.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms as a monocyclic ring or 7to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycles having 7 to12 atoms can be arranged, for example, as a bicyclo[4,5], [5,5], [5,6]or [6,6] system, and bicyclic carbocycles having 9 or 10 ring atoms canbe arranged as a bicyclo[5,6] or [6,6] system, or as bridged systemssuch as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like.

The terms “heterocycle,” “heterocyclyl” and “heterocyclic ring” are usedinterchangeably herein and refer to a saturated or a partiallyunsaturated (i.e., having one or more double and/or triple bonds withinthe ring) carbocyclic radical of 3 to 20 ring atoms in which at leastone ring atom is a heteroatom selected from nitrogen, oxygen and sulfur,the remaining ring atoms being C, where one or more ring atoms isoptionally substituted independently with one or more substituentsdescribed below. A heterocycle may be a monocycle having 3 to 7 ringmembers (2 to 6 carbon atoms and 1 to 4 heteroatoms selected from N, O,P, and S) or a bicycle having 7 to 10 ring members (4 to 9 carbon atomsand 1 to 6 heteroatoms selected from N, O, P, and S), for example: abicyclo[4,5], [5,5], [5,6], or [6,6] system. Heterocycles are describedin Paquette, Leo A.; “Principles of Modern Heterocyclic Chemistry” (W.A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and9; “The Chemistry of Heterocyclic Compounds, A series of Monographs”(John Wiley & Sons, New York, 1950 to present), in particular Volumes13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566. The term“heterocycle” includes heterocycloalkoxy. “Heterocyclyl” also includesradicals where heterocycle radicals are fused with a saturated,partially unsaturated ring, or aromatic carbocyclic or heterocyclicring. Examples of heterocyclic rings include, but are not limited to,pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl,tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino,morpholino, thiomorpholino, thioxanyl, piperazinyl, homopiperazinyl,azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl,oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl,indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl,dihydrofuranyl, pyrazolidinylimidazolinyl, imidazolidinyl,3-azabicyco[3.1.0]hexanyl, 3-azabicyclo[4.1.0]heptanyl,azabicyclo[2.2.2]hexanyl, 3H-indolyl quinolizinyl and N-pyridyl ureas.Spiro moieties are also included within the scope of this definition.Examples of a heterocyclic group wherein 2 ring carbon atoms aresubstituted with oxo (═O) moieties are pyrimidinonyl and1,1-dioxo-thiomorpholinyl. The heterocycle groups herein are optionallysubstituted independently with one or more substituents describedherein.

“Aryl” means a monovalent aromatic hydrocarbon radical of 6-20 carbonatoms derived by the removal of one hydrogen atom from a single carbonatom of a parent aromatic ring system. Some aryl groups are representedin the exemplary structures as “Ar”. Aryl includes bicyclic radicalscomprising an aromatic ring fused to a saturated, partially unsaturatedring, or aromatic carbocyclic or heterocyclic ring. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene (phenyl),substituted benzenes, naphthalene, anthracene, biphenyl, indenyl,indanyl, 1,2-dihydronapthalene, 1,2,3,4-tetrahydronapthyl, and the like.Aryl groups are optionally substituted independently with one or moresubstituents described herein.

The term “heteroaryl” refers to a monovalent aromatic radical of 5-, 6-,or 7-membered rings, and includes fused ring systems (at least one ofwhich is aromatic) of 5-20 atoms, containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur. Examples ofheteroaryl groups are pyridinyl (including, for example,2-hydroxypyridinyl), imidazolyl, imidazopyridinyl, pyrimidinyl(including, for example, 4-hydroxypyrimidinyl), pyrazolyl, triazolyl,pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl,isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl,benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl,phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl,oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furazanyl,benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl,quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl.Heteroaryl groups are optionally substituted independently with one ormore substituents described herein.

The heterocycle or heteroaryl groups may be carbon (carbon-linked),nitrogen (nitrogen-linked) or oxygen (oxygen-linked) attached where suchis possible. By way of example and not limitation, carbon bondedheterocycles or heteroaryls are bonded at position 2, 3, 4, 5, or 6 of apyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4,or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole ortetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole orthiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole,position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine,position 2, 3, 4, 5, 6, 7, or 8 of a quinoline or position 1, 3, 4, 5,6, 7, or 8 of an isoquinoline.

By way of example and not limitation, nitrogen bonded heterocycles orheteroaryls are bonded at position 1 of an aziridine, azetidine,pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole,imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline,2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline,1H-indazole, position 2 of a isoindole, or isoindoline, position 4 of amorpholine, and position 9 of a carbazole, or β-carboline.

“Carbon linked monocyclic heteroaryl” refers to a five- or six-membered,unsubstituted or substituted, monocyclic heteroaryl radical whichcontains 1, 2, 3 or 4 ring heteroatoms independently selected from N, Oand S. The carbon linked monocyclic heteroaryl is attached to the C-2position of the pyrimidine ring according to Formulas I at any carbonatom of the monocyclic heteroaryl R³ group. Carbon linked monocyclicheteroaryl radicals include, but are not limited to: 2-pyridyl,3-pyridyl, 4-pyridyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl,2-imidazolyl, 4-imidazolyl, 3-pyrazolyl, 4-pyrazolyl, 2-pyrrolyl,3-pyrrolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 2-pyrimidinyl, 5-pyrimidinyl,6-pyrimidinyl, 2-pyrazinyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl,2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 3-triazolyl, 1-triazolyl,5-tetrazolyl, 1-tetrazolyl, and 2-tetrazolyl. Carbon linked monocyclicheteroaryls are optionally substituted independently with one or moresubstituents described herein.

“Carbon linked fused bicyclic C₃-C₂₀ heterocyclyl” and “carbon linkedfused bicyclic C₁-C₂₀ heteroaryl” containing one or more heteroatomsindependently selected from nitrogen, oxygen, and sulfur, differ only bytheir aromatic character, and have two rings fused together, i.e. sharea common bond. Carbon linked fused bicyclic heterocyclyl and heteroarylradicals are attached to the C-2 position of the pyrimidine ringaccording to Formulas I at any carbon atom of the fused bicyclic C₃-C₂₀heterocyclyl or fused bicyclic C₁-C₂₀ heteroaryl group R³ group. Carbonlinked fused bicyclic heterocyclyl and heteroaryl radicals include, butare not limited to: 1H-indazole, 1H-indole, indolin-2-one,1-(indolin-1-yl)ethanone, 1H-benzo[d][1,2,3]triazole,1H-pyrazolo[3,4-b]pyridine, 1H-pyrazolo[3,4-d]pyrimidine,1H-benzo[d]imidazole, 1H-benzo[d]imidazol-2(3H)-one,1H-pyrazolo[3,4-c]pyridine, 1H-pyrrolo[2,3-c]pyridine,3H-imidazo[4,5-c]pyridine, 7H-pyrrolo[2,3-d]pyrimidine, 7H-purine,1H-pyrazolo[4,3-d]pyrimidine, 5H-pyrrolo[3,2-d]pyrimidine,2-amino-1H-purin-6(9H)-one, quinoline, quinazoline, quinoxaline,isoquinoline, isoquinolin-1(2H)-one, 3,4-dihydroisoquinolin-1(2H)-one,3,4-dihydroquinolin-2(1H)-one, quinazolin-2(1H)-one,quinoxalin-2(1H)-one, 1,8-naphthyridine, pyrido[3,4-d]pyrimidine, andpyrido[3,2-b]pyrazine. Fused bicyclic heterocycles and fused bicyclicheteroaryls are optionally substituted independently with one or moresubstituents described herein.

The substituent groups that alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, heteroaryl, fused bicyclic C₄-C₂₀ heterocyclyl, andfused bicyclic C₁-C₂₀ heteroaryl are optionally substituted with includeF, Cl, Br, I, CN, CF₃, —NO₂, oxo, R¹⁰, —C(═Y)R¹⁰, —C(═Y)OR¹⁰,—C(═Y)NR¹⁰R¹¹, —(CR¹⁴R¹⁵)_(n)NR¹⁰R¹¹, —(CR¹⁴R¹⁵)_(n)OR¹⁰, —NR¹⁰R¹¹,—NR¹²C(═Y)R¹⁰, —NR¹²C(═Y)OR¹¹, —NR¹²C(═Y)NR¹⁰R¹¹, —NR¹²SO₂R¹⁰, ═NR¹²,OR¹⁰, —OC(═Y)R¹⁰, —OC(═Y)OR¹⁰, —OC(═Y)NR¹⁰R¹¹, —OS(O)₂(OR¹⁰),—OP(═Y)(OR¹⁰)(OR¹¹), —OP(OR¹⁰)(OR¹¹), SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰,—S(O)₂NR¹⁰R¹¹, —S(O)(OR¹⁰), —S(O)₂(OR¹⁰), —SC(═Y)R¹⁰, —SC(═Y)OR¹⁰,—SC(═Y)NR¹⁰R¹¹, C₁-C₁₂ optionally substituted alkyl, C₂-C₈ optionallysubstituted alkenyl, C₂-C₈ optionally substituted alkynyl, C₃-C₁₂optionally substituted carbocyclyl, C₂-C₂₀ optionally substitutedheterocyclyl, C₆-C₂₀ optionally substituted aryl, C₁-C₂₀ optionallysubstituted heteroaryl, —(CR¹⁴R¹⁵)_(t)—NR¹²C(═O)(CR¹⁴R¹⁵)NR¹⁰R¹¹, and(CR⁴R⁵)_(t)—NR¹⁰R¹¹

A “metabolite” is a product produced through metabolism in the body of aspecified compound or salt thereof. Metabolites of a compound may beidentified using routine techniques known in the art and theiractivities determined using tests such as those described herein. Suchproducts may result for example from the oxidation, reduction,hydrolysis, amidation, deamidation, esterification, deesterification,enzymatic cleavage, and the like, of the administered compound.Accordingly, the invention includes metabolites of compounds of theinvention, including compounds produced by a process comprisingcontacting a compound of this invention with a mammal for a period oftime sufficient to yield a metabolic product thereof.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts.

The phrase “pharmaceutically acceptable salt” as used herein, refers topharmaceutically acceptable organic or inorganic salts of a compound ofthe invention. Exemplary salts include, but are not limited, to sulfate,citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucuronate, saccharate, formate, benzoate, glutamate,methanesulfonate “mesylate”, ethanesulfonate, benzenesulfonate,p-toluenesulfonate, and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. A pharmaceuticallyacceptable salt may involve the inclusion of another molecule such as anacetate ion, a succinate ion or other counter ion. The counter ion maybe any organic or inorganic moiety that stabilizes the charge on theparent compound. Furthermore, a pharmaceutically acceptable salt mayhave more than one charged atom in its structure. Instances wheremultiple charged atoms are part of the pharmaceutically acceptable saltcan have multiple counter ions. Hence, a pharmaceutically acceptablesalt can have one or more charged atoms and/or one or more counter ion.

If the compound of the invention is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,methanesulfonic acid, phosphoric acid and the like, or with an organicacid, such as acetic acid, maleic acid, succinic acid, mandelic acid,fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid,salicylic acid, a pyranosidyl acid, such as glucuronic acid orgalacturonic acid, an alpha hydroxy acid, such as citric acid ortartaric acid, an amino acid, such as aspartic acid or glutamic acid, anaromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid,such as p-toluenesulfonic acid or ethanesulfonic acid, or the like.Acids which are generally considered suitable for the formation ofpharmaceutically useful or acceptable salts from basic pharmaceuticalcompounds are discussed, for example, by P. Stahl et al, Camille G.(eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use.(2002) Zurich: Wiley-VCH; S. Berge et al, Journal of PharmaceuticalSciences (1977) 66(1) 1 19; P. Gould, International J. of Pharmaceutics(1986) 33 201 217; Anderson et al, The Practice of Medicinal Chemistry(1996), Academic Press, New York; Remington's Pharmaceutical Sciences,18^(th) ed., (1995) Mack Publishing Co., Easton Pa.; and in The OrangeBook (Food & Drug Administration, Washington, D.C. on their website).These disclosures are incorporated herein by reference thereto.

If the compound of the invention is an acid, the desiredpharmaceutically acceptable salt may be prepared by any suitable method,for example, treatment of the free acid with an inorganic or organicbase, such as an amine (primary, secondary or tertiary), an alkali metalhydroxide or alkaline earth metal hydroxide, or the like. Illustrativeexamples of suitable salts include, but are not limited to, organicsalts derived from amino acids, such as glycine and arginine, ammonia,primary, secondary, and tertiary amines, and cyclic amines, such aspiperidine, morpholine and piperazine, and inorganic salts derived fromsodium, calcium, potassium, magnesium, manganese, iron, copper, zinc,aluminum and lithium.

The phrase “pharmaceutically acceptable” indicates that the substance orcomposition must be compatible chemically and/or toxicologically, withthe other ingredients comprising a formulation, and/or the mammal beingtreated therewith.

The term “synergistic” as used herein refers to a therapeuticcombination which is more effective than the additive effects of the twoor more single agents. Determination of a synergistic interactionbetween a Formula I compound, and one or more chemotherapeutic agent maybe based on the results obtained from the assays described herein. Theresults of these assays are analyzed using the Chou and Talalaycombination method and Dose-Effect Analysis with CalcuSyn software inorder to obtain a Combination Index (Chou and Talalay, Trends Pharmacol.Sci. 4:450-454; Chou, T. C. (2006) Pharmacological Reviews68(3):621-681; Chou and Talalay, 1984, Adv. Enzyme Regul. 22:27-55). Thecombinations provided by this invention have been evaluated in severalassay systems, and the data can be analyzed utilizing a standard programfor quantifying synergism, additivism, and antagonism among anticanceragents. An exemplary program utilized is described by Chou and Talalay,in “New Avenues in Developmental Cancer Chemotherapy,” Academic Press,1987, Chapter 2. Combination Index values less than 0.8 indicatessynergy, values greater than 1.2 indicate antagonism and values between0.8 to 1.2 indicate additive effects. The combination therapy mayprovide “synergy” and prove “synergistic”, i.e., the effect achievedwhen the active ingredients used together is greater than the sum of theeffects that results from using the compounds separately. A synergisticeffect may be attained when the active ingredients are: (1)co-formulated and administered or delivered simultaneously in acombined, unit dosage formulation; (2) delivered by alternation or inparallel as separate formulations; or (3) by some other regimen. Whendelivered in alternation therapy, a synergistic effect may be attainedwhen the compounds are administered or delivered sequentially, e.g., bydifferent injections in separate syringes. In general, duringalternation therapy, an effective dosage of each active ingredient isadministered sequentially, i.e., serially, whereas in combinationtherapy, effective dosages of two or more active ingredients areadministered together.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®., polyethylene glycol (PEG), and PLURONICS®.

The term “therapeutically effective amount” refers to an amount of thetherapeutic combination effective to “treat” a disease or disorder in asubject or mammal. In the case of cancer, the therapeutically effectiveamount of the antagonist may reduce the number of cancer cells; reducethe tumor size; inhibit (i.e., slow to some extent and preferably stop)cancer cell infiltration into peripheral organs; inhibit (i.e., slow tosome extent and preferably stop) tumor metastasis; inhibit, to someextent, tumor growth; and/or relieve to some extent one or more of thesymptoms associated with the cancer. See the definition herein of“treating”. To the extent the antagonist may prevent growth and/or killexisting cancer cells, it may be cytostatic and/or cytotoxic.

A “growth inhibitory amount” of the therapeutic combination is an amountcapable of inhibiting the growth of a cell, especially tumor, e.g.,cancer cell, either in vitro or in vivo. A “growth inhibitory amount” ofthe therapeutic combination for purposes of inhibiting neoplastic cellgrowth may be determined empirically and in a routine manner.

A “cytotoxic amount” of the therapeutic combination is an amount capableof causing the destruction of a cell, especially tumor, e.g., cancercell, either in vitro or in vivo.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated increase in cell number (generally referred to herein ascell growth), which can be due to abnormal increase in cellproliferation, abnormal decrease of cell death, or an imbalance ofamounts of cell proliferation and cell death. Examples of cancerinclude, but are not limited to, hematopoietic cancers or blood-relatedcancers, such as lymphoma, leukemia, myeloma or lymphoid malignancies,but also cancers of the spleen and cancers of the lymph nodes.

The term “hyperproliferative” refers to disorders that are associatedwith some degree of abnormal cell proliferation. In one embodiment, ahyperproliferative disorder is cancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer”, “cancerous”, “cellproliferative disorder”, “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The term “non-Hodgkin's lymphoma” or “NHL”, as used herein, refers to acancer of the lymphatic system other than Hodgkin's lymphomas. Hodgkin'slymphomas can generally be distinguished from non-Hodgkin's lymphomas bythe presence of Reed-Sternberg cells in Hodgkin's lymphomas and theabsence of said cells in non-Hodgkin's lymphomas. Examples ofnon-Hodgkin's lymphomas encompassed by the term as used herein includeany that would be identified as such by one skilled in the art (e.g., anoncologist or pathologist) in accordance with classification schemesknown in the art, such as the Revised European-American Lymphoma (REAL)scheme as described in Color Atlas of Clinical Hematology, ThirdEdition; A. Victor Hoffbrand and John E. Pettit (eds.) (HarcourtPublishers Limited 2000) (see, in particular FIG. 11.57, 11.58 and/or11.59). More specific examples include, but are not limited to, relapsedor refractory NHL, front line low grade NHL, Stage III/IV NHL,chemotherapy resistant NHL, precursor B lymphoblastic leukemia and/orlymphoma, small lymphocytic lymphoma, B cell chronic lymphocyticleukemia and/or prolymphocytic leukemia and/or small lymphocyticlymphoma, hematopoietic prolymphocytic lymphoma, immunocytoma and/orlymphoplasmacytic lymphoma, marginal zone B cell lymphoma, splenicmarginal zone lymphoma, extranodal marginal zone—MALT lymphoma, nodalmarginal zone lymphoma, hairy cell leukemia, plasmacytoma and/or plasmacell myeloma, low grade/follicular lymphoma, intermediategrade/follicular NHL, mantle cell lymphoma, follicle center lymphoma(follicular), intermediate grade diffuse NHL, diffuse largehematopoietic lymphoma, aggressive NHL (including aggressive front-lineNHL and aggressive relapsed NHL), NHL relapsing after or refractory toautologous stem cell transplantation, primary mediastinal largehematopoietic lymphoma, primary effusion lymphoma, high gradeimmunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL, bulky disease NHL, Burkitt's lymphoma, precursor(peripheral) T-cell lymphoblastic leukemia and/or lymphoma, adult T-celllymphoma and/or leukemia, T cell chronic lymphocytic leukemia and/orprolymphocytic leukemia, large granular lymphocytic leukemia, mycosisfungoides and/or Sezary syndrome, extranodal natural killer/T-cell(nasal type) lymphoma, enteropathy type T-cell lymphoma, hepatosplenicT-cell lymphoma, subcutaneous panniculitis like T-cell lymphoma, skin(cutaneous) lymphomas, anaplastic large cell lymphoma, angiocentriclymphoma, intestinal T cell lymphoma, peripheral T-cell (not otherwisespecified) lymphoma and angioimmunoblastic T-cell lymphoma.

Non-Hodgkin's lymphoma thus includes hematopoietic lymphoma, B celllymphoma, diffuse large B cell lymphoma, follicular lymphoma, smalllymphocytic lymphoma, malignant lymphoma, malignant T cell lymphoma,anaplastic large cell lymphoma, and mucosal associated lymphoid tissuelymphoma.

Formula I Compounds

The present invention includes therapeutic combinations includingFormula I compounds which have the structures:

or stereoisomers, geometric isomers, tautomers, or pharmaceuticallyacceptable salts thereof, where:

R¹ is selected from H, F, Cl, Br, I, CN, —(CR¹⁴R¹⁵)_(m)NR¹⁰R¹¹,—C(R¹⁴R¹⁵)_(n)NR¹²C(═Y)R¹⁰, —(CR¹⁴R¹⁵)_(n)NR¹²S(O)₂R¹⁰,—(CR¹⁴R¹⁵)_(m)OR¹⁰, —(CR¹⁴R¹⁵)_(n)S(O)₂R¹⁰, —(CR¹⁴R¹⁵)_(n)S(O)₂NR¹⁰R¹¹,—C(OR¹⁰)R¹¹R¹⁴, —C(═Y)R¹⁰, —C(═Y)OR¹⁰, —C(═Y)NR¹⁰R¹¹, —C(═Y)NR¹²OR¹⁰,—C(═O)NR¹²S(O)₂R¹⁰, —C(═O)NR¹²(CR¹⁴R¹⁵)_(m)NR¹⁰R¹¹, —NO₂, —NR¹²C(═Y)R¹¹,—NR¹²C(═Y)OR¹¹, —NR¹²C(═Y)NR¹⁰R¹¹, —NR¹²S(O)₂R¹⁰, —NR¹²SO₂NR¹⁰R¹¹,—SR¹⁰, —S(O)₂R¹⁰, —S(O)₂NR¹⁰R¹¹, —SC(═Y)R¹⁰, —SC(═Y)OR¹⁰, C₁-C₁₂ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl,C₆-C₂₀ aryl, and C₁-C₂₀ heteroaryl;

R² is selected from H, F, Cl, Br, I, CN, CF₃, —NO₂, —C(═Y)R¹⁰,—C(═Y)OR¹⁰, —C(═Y)NR¹⁰R¹¹, —(CR¹⁴R¹⁵)_(m)NR¹⁰R¹¹, —(CR¹⁴R¹⁵)_(n)OR¹⁰,(CR¹⁴R¹⁵)_(t)—NR¹²C(═O)(CR¹⁴R¹⁵)NR¹⁰R¹¹, —NR¹²C(═Y)R¹⁰, —NR¹²C(═Y)OR¹⁰,—NR¹²C(═Y)NR¹⁰R¹¹, —NR¹²SO₂R¹⁰, OR¹⁰, —OC(═Y)R¹⁰, —OC(═Y)OR¹⁰,—OC(═Y)NR¹⁰R¹¹, —OS(O)₂(OR¹⁰), —OP(═Y)(OR¹⁰)(OR¹¹), —OP(OR¹⁰)(OR¹¹)SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —S(O)₂NR¹⁰R¹¹, —S(O)(OR¹⁰), —S(O)₂(OR¹⁰),—SC(═Y)R¹⁰, —SC(═Y)OR¹⁰, —SC(═Y)NR¹⁰R¹¹, C₁-C₁₂ alkyl, C₂-C₈ alkenyl,C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, andC₁-C₂₀ heteroaryl;

R³ is a carbon linked monocyclic heteroaryl, a carbon linked fusedbicyclic C₃-C₂₀ heterocyclyl, or a carbon linked fused bicyclic C₁-C₂₀heteroaryl, where the monocyclic heteroaryl, fused bicyclic C₃-C₂₀heterocyclyl, and fused bicyclic C₁-C₂₀ heteroaryl are optionallysubstituted with one or more groups selected from F, Cl, Br, I, —CN,—NR¹⁰R¹¹, —OR¹⁰, —C(O)R¹⁰, —NR¹⁰C(O)R¹¹, —N(C(O)R¹¹)₂, —NR¹⁰C(O)NR¹⁰R¹¹,—NR¹²S(O)₂R¹⁰, —C(═O)OR¹⁰, —C(═O)NR¹⁰R¹¹, C₁-C₁₂ alkyl and (C₁-C₁₂alkyl)-OR¹⁰;

R¹⁰, R¹¹ and R¹² are independently H, C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, or C₁-C₂₀heteroaryl,

or R¹⁰ and R¹¹ together with the nitrogen to which they are attachedform a C₂-C₂₀ heterocyclic ring optionally substituted with one or moregroups independently selected from oxo, (CH₂)_(m)OR¹², NR¹²R¹², CF₃, F,Cl, Br, I, SO₂R¹², C(═O)R¹², NR¹²C(═Y)R¹², NR¹²S(O)₂R¹², C(═Y)NR¹²R¹²,C₁-C₁₂ alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀heterocyclyl, C₆-C₂₀ aryl and C₁-C₂₀ heteroaryl;

R¹⁴ and R¹⁵ are independently selected from H, C₁-C₁₂ alkyl, or—(CH₂)_(n)-aryl,

or R¹⁴ and R¹⁵ together with the atoms to which they are attached form asaturated or partially unsaturated C₃-C₁₂ carbocyclic ring;

where said alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl, are optionally substituted with one or more groupsindependently selected from F, Cl, Br, I, CN, CF₃, —NO₂, oxo, R¹⁰,—C(═Y)R¹⁰, —C(═Y)OR¹⁰, —C(═Y)NR¹⁰R¹¹, —(CR¹⁴R¹⁵)_(n)NR¹⁰R¹¹,—(CR¹⁴R¹⁵)_(n)OR¹⁰, —NR¹⁰R¹¹, —NR¹²C(═Y)R¹⁰, —NR¹²C(═Y)OR¹¹,—NR¹²C(═Y)NR¹⁰R¹¹, —(CR¹⁴R¹⁵)_(m)NR¹²SO₂R¹⁰, ═NR¹², OR¹⁰, —OC(═Y)R¹⁰,—OC(═Y)OR¹⁰, —OC(═Y)NR¹⁰R¹¹, —OS(O)₂(OR¹⁰), —OP(═Y)(OR¹⁰)(OR¹¹),—OP(OR¹⁰)(OR¹¹), —SR¹⁰, —S(O)R¹⁰, —S(O)₂R¹⁰, —S(O)₂NR¹⁰R¹¹, —S(O)(OR¹⁰),—S(O)₂(OR¹⁰), —SC(═Y)R¹⁰, —SC(═Y)OR¹⁰, —SC(═Y)NR¹⁰R¹¹, C₁-C₁₂ alkyl,C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl,C₆-C₂₀ aryl, and C₁-C₂₀ heteroaryl;

Y is O, S, or NR¹²;

m is 0, 1, 2, 3, 4, 5 or 6; and

n is 1, 2, 3, 4, 5 or 6.

Exemplary embodiments of Formula I compounds include wherein R¹ is—(CR¹⁴R¹⁵)_(m)NR¹⁰R¹¹ where m is 1, and R¹⁰ and R¹¹ together with thenitrogen to which they are attached form an optionally substitutedC₃-C₂₀ heterocyclic ring. The C₃-C₂₀ heterocyclic ring may bepiperazinyl, optionally substituted with one or more groups selectedfrom NR¹⁰R¹¹, CF₃, F, Cl, Br, I, SO₂R¹⁰, C(═O)R¹⁰, NR¹²C(═Y)R¹¹,NR¹²S(O)₂R¹¹, C(═Y)NR¹⁰R¹¹, and C₁-C₁₂ alkyl.

Exemplary embodiments of Formula I compounds include wherein R¹ is notH.

Exemplary embodiments of Formula I compounds include wherein R² is H,CH₃, C₃-C₁₂ carbocyclyl, C₂-C₂₀ heterocyclyl, C₆-C₂₀ aryl, or C₁-C₂₀heteroaryl. The C₁-C₂₀ heteroaryl may be a monocyclic heteroaryl groupselected from 2-pyridyl, 3-pyridyl, 4-pyridyl, 3-isoxazolyl,4-isoxazolyl, 5-isoxazolyl, 2-imidazolyl, 4-imidazolyl, 3-pyrazolyl,4-pyrazolyl, 2-pyrrolyl, 3-pyrrolyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 2-pyrimidinyl,5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 2-oxazolyl, 4-oxazolyl,5-oxazolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 3-triazolyl,1-triazolyl, 5-tetrazolyl, 1-tetrazolyl, and 2-tetrazolyl.

Exemplary embodiments of Formula I compounds include wherein R³ is2-aminopyrimidin-5-yl.

Exemplary embodiments of Formula I compounds include wherein R³ is abicyclic heteroaryl group selected from 1H-indazole, 1H-indole,indolin-2-one, 1-(indolin-1-yl)ethanone, 1H-benzo[d][1,2,3]triazole,1H-pyrazolo[3,4-b]pyridine, 1H-pyrazolo[3,4-d]pyrimidine,1H-benzo[d]imidazole, 1H-benzo[d]imidazol-2(3H)-one,1H-pyrazolo[3,4-c]pyridine, 1H-pyrrolo[2,3-c]pyridine,3H-imidazo[4,5-c]pyridine, 7H-pyrrolo[2,3-d]pyrimidine, 7H-purine,1H-pyrazolo[4,3-d]pyrimidine, 5H-pyrrolo[3,2-d]pyrimidine,2-amino-1H-purin-6(9H)-one, quinoline, quinazoline, quinoxaline,isoquinoline, isoquinolin-1(2H)-one, 3,4-dihydroisoquinolin-1(2H)-one,3,4-dihydroquinolin-2(1H)-one, quinazolin-2(1H)-one,quinoxalin-2(1H)-one, 1,8-naphthyridine, pyrido[3,4-d]pyrimidine, andpyrido[3,2-b]pyrazine.

Exemplary embodiments of Formula I compounds include wherein R³ is1H-indazol-4-yl.

An exemplary Formula I compound is named as4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine;registered as CAS Reg. No. 957054-30-7; described and claimed in US2008/0076768; disclosed in Folkes et al (2008) Jour. of Med. Chem.51(18):5522-5532; Belvin et al, American Association for Cancer ResearchAnnual Meeting 2008, 99th: April 15, Abstract 4004; Folkes et al,American Association for Cancer Research Annual Meeting 2008, 99th:April 14, Abstract LB-146; Friedman et al, American Association forCancer Research Annual Meeting 2008, 99th: April 14, Abstract LB-110;and has Formula Ia:

Another exemplary Formula I compound is named as(S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one;disclosed and claimed in US 2008/0242665; and has Formula Ib:

Preparation of Formula I Compounds

The Formula I compounds may be synthesized by synthetic routes thatinclude processes analogous to those well-known in the chemical arts.Starting materials are generally available from commercial sources suchas Aldrich Chemicals (Milwaukee, Wis.) or are readily prepared usingmethods well known to those skilled in the art (e.g., prepared bymethods generally described in Louis F. Fieser and Mary Fieser, Reagentsfor Organic Synthesis, v. 1-19, Wiley, N.Y. (1967-1999 ed.), orBeilsteins Handbuch der organischen Chemie, 4, Aufl. ed.Springer-Verlag, Berlin, including supplements (also available via theBeilstein online database).

Formula I compounds may be prepared using procedures to prepare otherthiophenes and pyrimidines (U.S. Pat. No. 6,608,053; U.S. Pat. No.6,492,383; U.S. Pat. No. 6,232,320; U.S. Pat. No. 6,187,777; U.S. Pat.No. 3,763,156; U.S. Pat. No. 3,661,908; U.S. Pat. No. 3,475,429; U.S.Pat. No. 5,075,305; US 2003/220365; GB 1393161; WO 93/13664); and otherheterocycles, which are described in: Comprehensive HeterocyclicChemistry, Editors Katritzky and Rees, Pergamon Press, 1984.

Formula I compounds may be converted into a pharmaceutically acceptablesalt, and a salt may be converted into the free base compound, byconventional methods. Formula I compounds may be therapeuticallyeffective as a free base or as a pharmaceutically acceptable salt,depending on the desired properties such as solubility, dissolution,hygroscopic nature, and pharmacokinetics. Examples of pharmaceuticallyacceptable salts include salts with inorganic acids such as hydrochloricacid, hydrobromic acid, hydroiodic acid, sulphuric acid, nitric acid andphosphoric acid; and organic acids such as methanesulfonic acid,benzenesulphonic acid, formic acid, acetic acid, trifluoroacetic acid,propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid,maleic acid, lactic acid, malic acid, tartaric acid, citric acid,ethanesulfonic acid, aspartic acid and glutamic acid. The salt may be amesylate, a hydrochloride, a phosphate, a benzenesulfonate or a sulfate.Salts may be mono-salts or bis-salts. For example, the mesylate salt maybe the mono-mesylate or the bis-mesylate.

Formula I compounds and salts may also exist as hydrates or solvates.

Protection of functional groups (e.g., primary or secondary amine) ofintermediates may be necessary in preparing Formula I compounds. Theneed for such protection will vary depending on the nature of the remotefunctionality and the conditions of the preparation methods. Suitableamino-protecting groups include acetyl, trifluoroacetyl,t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz) and9-fluorenylmethyleneoxycarbonyl (Fmoc). The need for such protection isreadily determined by one skilled in the art. For a general descriptionof protecting groups and their use, see T. W. Greene, Protective Groupsin Organic Synthesis, John Wiley & Sons, New York, 1991.

For illustrative purposes, Schemes 1-7 show general methods forpreparing the compounds of the present invention as well as keyintermediates. For a more detailed description of the individualreaction steps, see the Examples section below. Those skilled in the artwill appreciate that other synthetic routes may be used to synthesizethe inventive compounds. Although specific starting materials andreagents are depicted in the Schemes and discussed below, other startingmaterials and reagents can be easily substituted to provide a variety ofderivatives and/or reaction conditions. In addition, many of thecompounds prepared by the methods described below can be furthermodified in light of this disclosure using conventional chemistry wellknown to those skilled in the art.

Scheme 1

Scheme 1 shows a general method for preparation of the thienopyrimidineintermediates 53 from 2-carboxyester, 3-amino thiophene reagents 51,wherein Hal is Cl, Br, or I; and R¹, R², and R¹⁰ are as defined forFormula I compounds, or precursors or prodrugs thereto.

Scheme 2

Scheme 2 shows a general method for selectively displacing a 4-halidefrom bis-halo thienopyrimidine intermediates 54 with morpholine underbasic conditions in an organic solvent to prepare 2-halo, 4-morpholinothienopyrimidine compounds 55, wherein Hal is Cl, Br, or I; and R¹ andR² are as defined for Formula I compounds, or precursors or prodrugsthereto.

Scheme 3

Scheme 3 shows a general method for derivatizing the 6-position of2-halo, 4-morpholino, 6-hydrogen thienopyrimidine compounds 56 where R¹is H. Treating 56 with a lithiating reagent to remove the 6 positionproton, followed by adding an acylating reagent R¹⁰C(O)Z where Z is aleaving group, such as halide, NHS ester, carboxylate, or dialkylamino,gives 2-halo, 4-morpholino, 6-acyl thienopyrimidine compounds 57,wherein Hal is Cl, Br, or I; and R² and R¹⁰ are as defined for Formula Icompounds, or precursors or prodrugs thereto. An example of R¹⁰C(O)Z toprepare 6-formyl compounds (R¹⁰═H) is N,N′-dimethylformamide (DMF).

Scheme 4

Scheme 4 shows a general method for Suzuki-type coupling of a 2-halopyrimidine intermediates 58 with a monocyclic heteroaryl, fused bicyclicheterocyclyl or fused bicyclic heteroaryl boronate acid (R¹⁵═H) or ester(R¹⁵=alkyl) reagent to prepare the 2-substituted (Hy), 4-morpholinothienopyrimidine compounds 59 of Formula I wherein Hal is Cl, Br, or I;and R¹ and R² are as defined for Formula I compounds, or precursors orprodrugs thereto. For reviews of the Suzuki reaction, see: Miyaura etal. (1995) Chem. Rev. 95:2457-2483; Suzuki, A. (1999) J. Organomet.Chem. 576:147-168; Suzuki, A. in Metal-Catalyzed Cross-CouplingReactions, Diederich, F., Stang, P. J., Eds., VCH, Weinheim, Del.(1998), pp 49-97. The palladium catalyst may be any that is typicallyused for Suzuki-type cross-couplings, such as PdCl₂(PPh₃)₂, Pd(PPh₃)₄,Pd(OAc)₂, PdCl₂(dppf)-DCM, Pd₂(dba)₃/Pt—Bu)₃ (Owens et al (2003)Bioorganic & Med. Chem. Letters 13:4143-4145; Molander et al (2002)Organic Letters 4(11):1867-1870; U.S. Pat. No. 6,448,433).

Scheme 5

Scheme 5 shows a general method for the synthesis of alkynes 61, whichcan be used to prepare alkynylated derivatives of compounds 63.Propargylic amines 61 may be prepared by reaction of propargyl bromide60 with an amine of the formula R¹⁰R¹¹NH (wherein R¹⁰ and R¹¹ areindependently selected from H, alkyl, aryl and heteroaryl, or R¹⁰ andR¹¹ together with the nitrogen to which they are attached form aheterocyclic ring) in the presence of an appropriate base (Cs₂CO₃ or thelike). For reviews of alkynyl amines and related syntheses seeBooker-Milbum, K. I., Comprehensive Organic Functional GroupTransformations (1995), 2:1039-1074; and Viehe, H. G., (1967) Angew.Chem., Int. Ed. Eng., 6(9):767-778. Alkynes 61 may subsequently bereacted with intermediates 62 (X²=bromo or iodo) via Sonogashiracoupling, to provide compounds 63, wherein R² and R³ are as defined forFormula I compounds, or precursors or prodrugs thereto.

Scheme 6

Scheme 6 shows a general method for the synthesis of alkynes 65, whichcan be used to prepare alkynylated derivatives of compounds 66.Gem-dialkyl propargylic amines 65 may be prepared using methodsdescribed by Zaragoza et al (2004) J. Med. Chem., 47:2833. According toScheme 6, gem-dialkyl chloride 64 (R¹⁴ and R¹⁵ are independently methyl,ethyl or other alkyl group) can be reacted with an amine of the formulaR¹⁰R¹¹NH (wherein R¹⁰ and R¹¹ are independently selected from H, alkyl,aryl and heteroaryl, or R¹⁰ and R¹¹ together with the nitrogen to whichthey are attached form a heterocyclic ring) in the presence of CuCl andan appropriate base (e.g. TEA or the like) to provide the alkyne 65.Alkyne 65 can be reacted with intermediates 62 (via Sonogashiracoupling) to provide compounds 66, wherein R² and R³ are as defined forFormula I compounds, or precursors or prodrugs thereto.

Scheme 7

Scheme 7 shows a general scheme for the synthesis of alkynes 68, whichcan be used to prepare alkynylated derivatives of compounds 69.But-3-yn-1-amines 68 (wherein R¹⁴ and R¹⁵ are independently H, alkyl,aryl, heteroaryl, or R¹⁴ and R¹⁵ together with the carbon atom to whichthey are attached form a carbocyclic or heterocyclic ring) can beprepared from reaction of alkynes 67 (LG=tosylate or other leavinggroup) with an amine of the formula R¹⁰R¹¹NH (wherein R¹⁰ and R¹¹ areindependently selected from H, alkyl, aryl and heteroaryl, or R¹⁰ andR¹¹ together with the nitrogen to which they are attached form aheterocyclic ring) using the protocol described by Olomucki M. et al(1960) Ann. Chim. 5:845. Alkynes 68 can subsequently be reacted withintermediates 62 via Sonogashira coupling, according to the descriptionsprovided for Schemes 5 and 6 to provide compounds 69, respectively,wherein R² and R³ are as defined for Formula I compounds, or precursorsor prodrugs thereto.

A pharmaceutically acceptable salt of a thienopyrimidine compound ofFormula I may be prepared using conventional techniques. Typically theprocess comprises treating the thienopyrimidine of Formula I as definedabove with a suitable acid in a suitable solvent.

In the process of the invention as defined above, both the aminationstep and the Pd-mediated cross-coupling step take place underconventional conditions. The palladium catalyst may be any that istypically used for Suzuki-type cross-couplings, such as PdCl₂(PPh₃)₂.The reducing agent is typically a borohydride, such as NaBH(OAc)₃, NaBH₄or NaCNBH₄.

Chemotherapeutic Agents

Certain chemotherapeutic agents have demonstrated surprising andunexpected properties in combination with Formula I compounds ininhibiting cellular proliferation in vitro and in vivo. Suchchemotherapeutic agents include: dexamethasone, thioTEPA, doxorubicin,vincristine, rituximab, cyclophosphamide, prednisone, melphalan,lenalidomide, bortezomib, rapamycin, and cytarabine.

Dexamethasone is a potent glucocorticoid steroid hormone, withanti-inflammatory and immunosuppressant activity. In oncology,dexamethasone is given to cancer patients undergoing chemotherapy, tocounteract certain side-effects of their antitumor treatment.Dexamethasone can augment the antiemetic effect of 5-HT₃ receptorantagonists like ondansetron. Dexamethasone is also used in certainhematological malignancies, especially in the treatment of multiplemyeloma, in which dexamethasone is given alone or together withthalidomide (thal-dex) or a combination of Adriamycin (doxorubicin) andvincristine (VAD). In brain tumours (primary or metastatic),dexamethasone is used to counteract the development of edema, whichcould eventually compress other brain structures. Dexamethasone is namedas(8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl-6,7,8,11,12,14,15,16-octahydrocyclopenta[a]phenanthren-3-one(CAS Reg. No. 50-02-2) and has the structure:

thioTEPA (tespa, thiophosphoamide, tespamin, tspa, tifosyl, THIOPLEX®)is an alkylating chemotherapeutic agent used to treat breast cancer,ovarian cancer, and bladder cancer (Maanen et al (2000) Cancer Treat Rev26(4):257-68; U.S. Pat. No. 2,670,347). It is also used as conditioningfor bone marrow transplantation ThioTEPA is named asN,N′N′-triethylenethiophosphoramide, phosphinothioylidynetrisaziridine,or 1,1′,1″-phosphorothioyltriaziridine (CAS Reg. No. 52-24-4) and hasthe structure:

Doxorubicin (ADRIAMYCIN®, hydroxyldaunorubicin) is a DNA-interactingdrug widely used in chemotherapy since the 1960s. It is an anthracyclineantibiotic and structurally related to daunomycin, which alsointercalates DNA. Doxorubicin is commonly used in the treatment of awide range of cancers. Doxorubicin is named as(8S,10S)-10-(4-amino-5-hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7,8,9,10-tetrahydrotetracene-5,12-dione,(CAS Reg. No. 23214-92-8) and has the structure:

Vincristine (22-Oxovincaleukoblastine; leurocristine, VCR, LCR sulfateform: Vincristine sulfate, Kyocristine, ONCOVIN® (Lilly), Vincosid,Vincrex), is a vinca alkaloid from the Madagascar periwinkleCatharanthus roseus, formerly Vinca rosea (Johnson et al (1963) CancerRes. 23:1390-1427; Neuss et al (1964) J. Am. Chem. Soc. 86:1440). Alongwith semisynthetic derivatives, vindesine and vinorelbine (NAVELBINE®,vincristine inhibits mitosis in metaphase by binding to tubulin andpreventing the cell from making spindles necessary to move chromosomesas the cell divides. Vincristine is a chemotherapy drug that is given asa treatment for some types of cancer including leukemia, lymphoma,breast and lung cancer. Vincristine (leurocristine, VCR) is mosteffective in treating childhood leukemias and non-Hodgkin's lymphomas,where vinblastine (vincaleukoblastine, VLB) is used to treat Hodgkin'sdisease. Vincristine (CAS number 57-22-7) has the structure:

Rituximab (RITUXAN®, Genentech/Biogen Idec; MABTHERA®, Roche, REDITUX®,CAS Reg. No. 174722-31-7) is a genetically engineered chimericmurine/human monoclonal antibody directed against the CD20 antigen.Rituximab is the antibody called “C2B8” in U.S. Pat. No. 5,736,137.Rituximab is indicated for the treatment of patients with relapsed orrefractory low-grade or follicular, CD20-positive, B-cell NHL. Rituximabbinds to cell surface CD-20 and results in B-cell depletion (Cartron etal (2002) Blood 99: 754-758; Idusogie et al (2000) J. Immunol. 164:4178-4184; Grillo-López A J, et al (1999) Semin Oncol; 26:66-73; U.S.Pat. No. 5,736,137). RITUXAN (U.S. Pat. No. 5,677,180; U.S. Pat. No.5,736,137) is the most widely used monoclonal antibody in hematopoieticmalignancies and is established in widespread clinical practice. RITUXANfirst received FDA approval in 1997 for the treatment of relapsed orrefractory, low-grade or follicular, CD20-positive, B-cell non-Hodgkin'slymphoma (NHL). It was also approved in the European Union under thetrade name MabThera® in June 1998. In February 2006, RITUXAN alsoreceived FDA approval in combination with methotrexate to reduce signsand symptoms in adult patients with moderately-to-severely-activerheumatoid arthritis who have had an inadequate response to one or moreTNF antagonist therapies. The amino acid sequence of rituximab antibody(also designated C2B8) and exemplary methods for its production viarecombinant expression in Chinese Hamster Ovary (CHO) cells aredisclosed in U.S. Pat. No. 5,736,137.

Cyclophosphamide (Cytoxan, Neosar, Revimmune, cyclophosphane, B-518,Cycloblastin, Cyclostin, Endoxan, Procytox, Sendoxan, cytophosphane) isa nitrogen mustard alkylating agent, from the oxazophorines group usedto treat various types of cancer and some autoimmune disorders (“AReview of Cyclophosphamide”, D. L. Hill (1975) Charles C. Thomas,Springfield, 340 pp; IARC Monographs (1975) 9:135-156; Fraiser et al(1991) Drugs 42:781-795; Colvin, OmM. (1999) Curr. Pharmaceut. Design5:555-560). Cyclophosphamide is a prodrug converted in the liver toactive forms that have chemotherapeutic activity. The main use ofcyclophosphamide is together with other chemotherapy agents in thetreatment of lymphomas, some forms of leukemia, and some solid tumors(Shanafelt et al (2007) Cancer 109(11): 2291-8; Brock N (1996) Cancer78(3):542-7). It is a chemotherapy drug that works by slowing orstopping cell growth and by decreasing the immune system response tovarious diseases.

Cyclophosphamide is named asN,N-bis(2-chloroethyl)-1,3,2-oxazaphosphinan-2-amine 2-oxide,N,N-bis(2-chloroethyl)tetrahydro-2H-1,3,2-oxazaphosphorin-2-amine-2-oxide;1-bis(2-chloroethyl)amino-1-oxo-2-aza-5-oxaphosphoridin monohydrate;bis(2-chloroethyl)-phosphamide cyclic propanolamide ester; orN,N-bis(beta-chloroethyl)-N′,O-propylenephosphoric acid ester diamide,including hydrate forms (CAS number 50-18-0), and has the structure:

Prednisone (Meticorten, Sterapred, Sterapred DS, retrocortine, Colisone,Cortancyl, Dacortin, Decortin, Deltacortene, Deltacortone, Deltasone,Deltison, Di-Adreson, Encorton, Hostacortin, Meticorten, Orasone,Rectodelt, Sone, or Ultracorten) is a synthetic corticosteroid drug(U.S. Pat. No. 2,897,216; U.S. Pat. No. 2,837,464; U.S. Pat. No.3,134,718; U.S. Pat. No. 2,579,479). Prednisone is a prodrug convertedin the liver into prednisolone (CAS Reg. No. 50-24-8), an 11-hydroxylanalog, and has a mainly glucocorticoid effect. Prednisone may beadministered orally or by injection. Prednisone is particularlyeffective as an immunosuppressant and is used to treat autoimmunediseases, inflammatory diseases (such as severe asthma, allergies,poison ivy, dermatitis, lupus, rheumatoid arthritis, and Crohn'sdisease, and to prevent and treat rejection in organ transplantation.Prednisone is used to treat cancer, including acute lymphoblasticleukemia, Non-Hodgkin's lymphomas, and multiple myeloma. Prednisone isnamed as17-hydroxy-17-(2-hydroxyacetyl)-10,13-dimethyl-7,8,9,10,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,11-dione;or 17,21-dihydroxypregna-1,4-diene-3,11,20-trione;1,4-pregnadiene-17alpha,21-diol-3,11,20-trione; (CAS number 53-03-2),and has the structure:

Melphalan (L-phenylalanine mustard; alanine nitrogen mustard; L-PAM;melfalan; L-sarcolysine; NSC-8806; CB-3025; ALKERAN® (Glaxo SmithKline);Sarcoclorin) is a nitrogen mustard alkylating agent type ofchemotherapeutic (U.S. Pat. No. 3,032,584; U.S. Pat. No. 3,032,585).Melphalan is used primarily to treat multiple myeloma, ovarian cancerand melanoma (IARC Monographs (1975) 9:167-180; Furner et al (1980)Cancer Treat. Rep. 64:559-574). Melphalan is named as2-amino-3-[4-[bis(2-chloroethyl)amino]phenyl]-propanoic acid;4-[bis(2-chloroethyl)amino]-L-phenylalanine; orp-di(2-chloroethyl)amino-L-phenylalanine (CAS Reg. No. 148-82-3) and hasthe structure:

Lenalidomide (REVLIMID®, CC5013, Revimid, Celgene Inc.) is a derivativeof thalidomide and introduced in 2004 (U.S. Pat. No. 5,635,517, U.S.Pat. No. 6,281,230) to treat both inflammatory disorders and cancers.There are multiple mechanisms of action, including a direct anti-tumoreffect, inhibition of the microenvironment support for tumor cells, andan immunomodulatory role. In vitro, lenalidomide induces tumor cellapoptosis directly and indirectly by inhibition of bone marrow stromalcell support, by anti-angiogenic and anti-osteoclastogenic effects, andby immunomodulatory activity. Lenalidomide was initially intended as atreatment for multiple myeloma, for which thalidomide is an acceptedtherapeutic modality, but has also shown efficacy in the class ofhematological disorders known as myelodysplastic syndromes (Richardsonet al (2002) Blood 100:3063; Bartlett et al (2004) Nature Rev.4:314-322; Mitsiades et al (2004) Curr. Opin. Invest. Drugs 5:635-647;Armoiry et al. (2008) J of Clin Pharmacy & Therapeutics 33:219-226; Listet al (2005) N. Engl. Jour. Med. 352:549-57). Lenalidomide is named as3-(4-amino-1-oxoisoindolin-2-yl)piperidine-2,6-dione;3-(4-amino-1,3-dihydro-1-oxo-2H-isoindol-2-yl)-2,6-piperidinedione;1-oxo-2-(2,6-dioxopiperidin-3-yl)-4-aminoisoindoline (CAS Reg. No.191732-72-6) and has the structure:

Bortezomib (MG-341, PS-341, VELCADE®, Millenium Pharm.) is a boronicacid proteasome inhibitor approved in the US for treating relapsedmultiple myeloma and mantle cell lymphoma. (WO 96/13266; U.S. Pat. No.5,780,454; U.S. Pat. No. 6,083,903; U.S. Pat. No. 6,297,217; U.S. Pat.No. 6,617,317; U.S. Pat. No. 6,713,446; U.S. Pat. No. 6,747,150; U.S.Pat. No. 6,958,319; U.S. Pat. No. 7,119,080). The boron atom inbortezomib binds the catalytic site of the 26S proteasome with highaffinity and specificity. In normal cells, the proteasome regulatesprotein expression and function by degradation of ubiquitinylatedproteins, and also cleanses the cell of abnormal or misfolded proteins.(Adams et al (2004) Cancer Invest 22(2):304-11; Bonvini (2007). Leukemia21(4):838-42). Bortezomib is named as[(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronicacid;(R)-3-methyl-1-((S)-3-phenyl-2-(pyrazine-2-carboxamido)propanamido)butylboronicacid; or[(1R)-3-methyl-1-[[(2S)-1-oxo-3-phenyl-2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl]-boronicacid (CAS Reg. No. 179324-69-7) and has the structure:

Rapamycin (sirolimus, RAPAMUNE®) is an immunosuppressant drug used toprevent rejection in organ transplantation, and is especially useful inkidney transplants. Rapamycin is a macrolide antibiotic produced by thebacterium Streptomyces hygroscopicus in a soil sample obtained from anisland called Rapa Nui, better known as Easter Island (Pritchard D I(2005). Drug Discovery Today 10 (10): 688-691). Rapamycin inhibits theresponse to interleukin-2 (IL-2) and thereby blocks activation of T- andhematopoietics. The mode of action of rapamycin is to bind the cytosolicprotein FK-binding protein 12 (FKBP12). The rapamycin-FKBP12 complexinhibits the mammalian target of rapamycin (mTOR) pathway throughdirectly binding the mTOR Complex1 (mTORC1). mTOR is also called FRAP(FKBP-rapamycin associated protein) or RAFT (rapamycin and FKBP target).Rapamycin analogs (“Rapalogs”) include Temsirolimus (CCI-779, Wyeth),Everolimus (RAD001, Novartis), Deforolimus (AP23573, MK-8669, Ariad,Merck). Rapamycin is named as(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]-oxaazacyclohentriacontine-1,5,11,28,29(4H,6H,31H)-pentone(CAS Reg. No. 53123-88-9), and has the structure:

Cytarabine (cytosine arabinoside, Ara-C, CYTOSAR-U®, Upjohn) is usedprimarily in the treatment of hematological malignancies, includingacute myeloid leukemia (AML) and NHL (U.S. Pat. No. 3,116,282; Shen etal (1965) J. Org. Chem. 835); Capizzi, R. L. (1996) Invest. New Drugs14:249-256; Grant S. (1998) Adv. Cancer Res. 72:197-233). Cytarabine isnamed as4-amino-1-((2R,3S,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one;4-amino-1-beta-D-arabinofuranosyl-2(1H)-pyrimidinone;1-beta-D-arabinofuranosylcytosine; (CAS Reg. No. 147-94-4) and has thestructure:

CHOP is an acronym for a chemotherapy regimen used in the treatment ofnon-Hodgkin lymphoma comprising cyclophosphamide, doxorubicin,vincristine, and prednisone/prednisolone (Fisher et al (1993) N Engl JMed 328(14):1002-6). CHOP is commonly administered in cycles of 4 weeks.A common treatment regimen is for at least 6 cycles.

Biological Evaluation

Certain Formula I compounds bind specifically to PI3 kinase isoforms andinhibit the proliferation of tumor cells (US 2008/0207611; US2008/0039459; US 2008/0076768; US 2008/0076758; US 2008/0242665; US2008/0269210). Certain exemplary Formula I compounds have PI3K bindingactivity IC₅₀ values less than 10 nM. Certain Formula I compounds havetumor cell-based activity EC₅₀ values less than 100 nM.

Certain exemplary therapeutic combinations of Formula I compounds andchemotherapeutic agents described herein were assayed for in vitroactivity against tumor cells (Example 15). Certain Formula I compoundsbind the p110α isoform at IC50 less than 1 micromole and showsingle-agent in vivo tumor growth inhibition in mouse xenograft models.Accordingly, Formula I compounds may be used to treat a disease ordisorder arising from abnormal cell growth, function or behavior assingle agents or in combination therapy with one or morechemotherapeutic agents.

Mutations in KRAS, NRAS, BRAF and PIK3CA activate two of the majorpathways mediating proliferation and anti-apoptotic signaling in cancercells. As such, mutations in these genes might constitute companiondiagnostic tests for targeted agents that inhibit key nodes in thesepathways, since the presence of a mutation may serve as a sign ofpathological activation and dependence on a given pathway in aparticular tumor. The mutation status for these genes, and others, in alarge panel of cell lines of diverse tissues of origin may yield acorrelation with response to selective inhibitors of MEK and PI3 kinase.In addition, mutation detection may be conducted on clinical samplesconsisting of small amounts of heterogeneous fixed tumor tissues, whichmay be analyzed using allele specific Taqman assays for the mostprevalent substitutions in KRAS, NRAS, BRAF and PI3 kinase.

FIGS. 1a and 1b shows reduction of pharmacodynamic (PD) markers measuredby FACS flow cytometry with Formula Ia (GDC-0941) treated (right column)and untreated (left column) cells, DoHH2 (lymphoma cells), WSU-DLCL2(lymphoma cells), OPM2 (multiple myeloma cells), and U266 (multiplemyeloma cells). Example 18 provides a FACS protocol for intracellulardetection of phospho-AKT (p-Akt) and p-S6 ribosomal protein (p-S6RP)post GDC-0941 treatment. Cells were treated in vitro with 5 μM GDC-0941for 4 hrs. Three of the cell lines showed evidence of PI3K pathwayactivation as evidenced by high levels of p-AKT and all four showevidence of distal pathway activation as evidenced by high levels ofphospho-S6 ribosomal protein signal in untreated cells (left columns).In the right column, cells treated with GDC-0941 have abolished p-AKTsignal and reduced or abolished p-S6RP signal. The remaining signals forpS6rp are consistent with a model that PI3k activity is partlyresponsible for this phosphorylation event. Collectively, these dataindicate that the PI3k pathway is activated in these cell types and thatGDC-0941 has potent inhibitory activity on the PI3k pathway in intactcells.

FIG. 2 shows reduction of pharmacodynamic (PD) markers p-AKT, p-S6RP,p-Bad, and in cells DoHH2, WSU-DLCL2, OPM2, and U266 as measured bySDS-polyacrylamide gel electrophoresis and western blotting in celllines treated in vitro with 5 μM GDC-0941 for 4 hrs. Example 17 providesa protocol for detection by Western blotting of p-Akt, p-BAD and p-S6ribosomal protein post GDC-0941 treatment of B cell and myeloma celllines. Cells were treated as indicated and lysates analyzed by WesternBlotting. Beta Actin blotting indicates approximately equal loading oflysates in each lane. Three of the cell lines showed evidence of PI3Kpathway activation as evidenced by high levels of p-AKT and all fourshow evidence of distal pathway activation as evidenced by high levelsof p-S6RP signal in untreated cells. Signals for both p-AKT and p-S6RPwere significantly reduced (where present) by treatment with GDC-0941indicating that the PI3K pathway has been activated in these cells andthat GDC-0941 has significant inhibitory activity on the pathway inintact cells. Levels of both PD markers follow the same rank order andare well correlated between FIGS. 1a and 1b and FIG. 2.

Pharmacodynamic and pharmacokinetic properties of absorption,distribution, metabolism, and excretion (ADME) were measured for certainexemplary compounds by assays including: Caco-2 Permeability, HepatocyteClearance, Cytochrome P450 Inhibition, Cytochrome P450 Induction, PlasmaProtein Binding, and hERG channel blockage.

The invention includes a method for determining compounds to be used incombination for the treatment of cancer comprising: a) administering atherapeutic combination of a compound having Formula I, and achemotherapeutic agent to an in vitro tumor cell line and, b) measuringa synergistic or non-synergistic effect.

In Vitro Cell Proliferation Assays

The cytotoxic or cytostatic activity of Formula I exemplary compoundswas measured by: establishing a proliferating mammalian tumor cell linein a cell culture medium, adding a Formula I compound, culturing thecells for a period from about 6 hours to about 5 days; and measuringcell viability (Example 15). Cell-based in vitro assays were used tomeasure viability, i.e. proliferation (IC₅₀), cytotoxicity (EC₅₀), andinduction of apoptosis (caspase activation).

The in vitro potency of the combinations of Formula I compounds withchemotherapeutic agents was measured by the cell proliferation assay ofExample 15; the CellTiter-Glo® Luminescent Cell Viability Assay,commercially available from Promega Corp., Madison, Wis. Thishomogeneous assay method is based on the recombinant expression ofColeoptera luciferase (U.S. Pat. No. 5,583,024; U.S. Pat. No. 5,674,713;U.S. Pat. No. 5,700,670) and determines the number of viable cells inculture based on quantitation of the ATP present, an indicator ofmetabolically active cells (Crouch et al (1993) J. Immunol. Meth.160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay wasconducted in 96 or 384 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons. Theextended half-life eliminates the need to use reagent injectors andprovides flexibility for continuous or batch mode processing of multipleplates. This cell proliferation assay can be used with various multiwellformats, e.g. 96 or 384 well format. Data can be recorded by luminometeror CCD camera imaging device. The luminescence output is presented asrelative light units (RLU), measured over time.

The anti-proliferative effects of Formula I exemplary compounds andcombinations with chemotherapeutic agents were measured by theCellTiter-Glo® Assay (Example 15) against the tumor cell lines in FIGS.3-6. EC₅₀ values were established for the tested compounds andcombinations. The range of in vitro cell potency activities was about100 nM to about 10 μM.

The individual measured EC50 values of the Formula I compounds and ofthe chemotherapeutic agent against the particular cell are compared tothe combination EC50 value. The combination index (CI) score iscalculated by the Chou and Talalay method (Chou, T. and Talalay, P.(1984) Adv. Enzyme Regul. 22:27-55). A CI less than 0.8 indicatessynergy. A CI between 0.8 and 1.2 indicates additivity. A CI greaterthan 1.2 indicates antagonism. The strength of synergy is assessedaccording to Chou and Talalay. Certain therapeutic combinations in FIGS.4-6 show the surprising and unexpected property of synergy in the invitro cell proliferation assays with tumor type cell lines includingnon-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), andmultiple myeloma. Other combinations show no synergy; and only show mereadditivity or antagonism. Certain combinations are synergistic with oneor more tumor types, but not others. The synergy demonstrated in the invitro cell proliferation assays provides a basis to expect acorresponding synergy in treating hematopoietic cancers including, butnot limited to, lymphoma and multiple myeloma in human patients.

FIG. 3 shows the effect of PI3K single agent inhibitor, Formula Ia(GDC-0941), and in combinations with dexamethasone (Dex) and doxorubicin(Dox), on B-NHL cell line DoHH2. In vitro cell survival andproliferation assays (Cell-Titer Glo, Promega) measured viable cellsover varying inhibitor concentrations (10⁻⁵ to 10 Relative Units of thepreviously (approximately) determined IC50, Formula Ia, dexamethasone,doxorubicin and combinations of Formula Ia and dexamethasone; andFormula Ia and doxorubicin. Note that the extent of cell killing at thehighest concentration of inhibitors varied from one agent to another. Inthe case of Doxorubicin, the addition of GDC-0941 caused a modest shiftof the dose-response curve to the left, indicating an increasedsensitivity of cells to the combination treatment. A combination index(CI) of ˜0.75 was calculated for this combination, indicating additivityor synergy. The relatively impotent activity of dexamethasone singleagent is reflected as a higher IC50 value as well as weaker extend ofsignal reduction in the assay, perhaps pointing to a cytostatic effector only weak cytotoxic activity. In combination with GDC-0941 however, asurprising and highly significant leftward shift of the dose-responsecurve was obtained at all concentrations. The calculated CI value at theIC50 point was ˜0.3, indicating an unpredicted and unexpectedly strongsynergy rarely seen between test agents.

Formula Ia (GDC-0941) is cytotoxic to many B lymphoma cell lines,inducing robust apoptosis as a single agent, according to Table 2.

TABLE 2 Cell Lines Tumor type EC50 (μM) SUDHL6 DLBCL 0.01 RI1 B-NHL 0.04SUDHL5 DLBCL 0.08 WSUDLCL2 DLBCL 0.13 MC116 NHL 0.16 WSUNHL NHL 0.16Rec1 B-NHL 0.16 Granta519 MCL 0.21 Farage DLBCL 0.24 DoHH2 DLBCL/FLL0.26 OciLy19 DLBCL 0.32 Jeko1 MCL 0.32 Bjab Burkit's 0.33 SUDHL4 DLBCL1.35 HT DLBCL 2.84 DB DLBCL 10 Ramos Burkit's 10 SC1 DLBCL 10 ToledoDLBCL 10

Data in table 2 indicate that GDC-0941 is broadly cytotoxic and potentagainst lymphoma cell lines at doses that could be clinicallyattainable.

Experiments such as those in FIG. 2 were extended to additional celllines and for combinations of GDC-0941 with thioTEPA, doxorubicin,vincristine, and dexamethasone. Combination Index (CI) scores werecalculated by the Chou-Talalay method (Table 3). In no case did we findthat CI values>1 which would indicate that GDC-0941 antagonized theseother agents when tested in combination. In general, GDC-0941 combinewell with these other agents showing at least additivity. Forcombinations with Dexamethasone, surprising and highly significant CIvalues of approximately 0.3 or less were obtained in all cell linestested.

TABLE 3 EC50 (μM) CI at CI at CI at CI at B-NHL Cell GDC-0941 EC50 EC50EC50 EC50 Lines (single agent) thioTEPA Dox vincristine Dex Bjab 0.330.88 0.64 0.83 0.30 DoHH2 0.16 0.65 0.75 Not 0.31 determined WSU-DLCL20.17 0.69 0.81 Not 0.12 determined

FIG. 4 shows the effect of PI3K single agent inhibitors on primaryfollicular lymphoma cells from patient NHL600 as determined by in vitrocell survival and proliferation assays (Cell-Titer Glo®, Promega Corp.,Madison, Wis.) measuring viable cells over varying concentrations (10⁻⁵to 10 μMolar) of Formula Ia, GDC-0464, and LY294002. As cell linecytoxicity data are commonly held to overestimate the potency of thesecompounds, the data indicate that Formula Ia (GDC-0941) has a surprisingand unexpected degree of potency against primary human cancer cells.GDC-0464 (Genentech, Inc.) is a potent thienopyrimidine PI3K inhibitor(US 2008/0076758). LY294002 (Eli Lilly & Co., CAS Reg. No. 154447-36-6)is also a potent inhibitor of PI3 kinases (WO 2003/035099).

FIG. 5 shows the effect of a PI3K single agent inhibitor, Formula Ia(GDC-0941), and in combination with doxorubicin, on primary diffuselarge B-cell lymphoma (DLBCL) cells from patient NHL640-A055. Cellviability was measured by in vitro cell survival and proliferationassays (Cell-Titer Glo®) over varying concentrations (10⁻⁵ to 20 Molar)of Formula Ia, doxorubicin, and the combination of Formula Ia anddoxorubicin. Anthracylines are the backbone of most chemotherapeuticregimens and as such are considered highly active compounds. Theseresults indicate that for this primary tumor sample in vitro, thatGDC-0941 was significantly more potent than doxorubicin and insurprising contrast to the results obtained with cell lines in vitro,that the combination was not significantly better than single agentGDC-0941.

FIG. 6 shows the effect of PI3K single agent inhibitor Formula Ia(GDC-0941), and in combination with dexamethasone, on multiple myelomaOPM2 cells by in vitro cell proliferation assays (Cell-Titer Glo®)measuring viable cells over varying drug concentrations (expressed as afunction of their previously determined IC50 values, i.e., “1”=[drug]giving an IC50 response) of Formula Ia, dexamethasone (Dex), and thecombination of Formula Ia and dexamethasone at fixed ratios. Therelatively weak response to dexamethasone is greatly enhanced bycombination with GDC-0941, both in terms of potency as well as theextent of response, and a CI value of 0.45 is obtained indicating strongsynergy between the two agents. Table 4 shows the Combination Indexscores, calculated by the Chou & Talalay method, of treatment of variousmultiple myeloma cell lines by the therapeutic combinations of FormulaIa compound (GDC-0941) and a chemotherapeutic agent selected fromdexamethasone (Dex), doxorubicin (Dox), melphalan, lenalidomide, andbortezomib. Certain combinations show synergy (CI<0.8), additivity(0.8-1.2), or antagonism (>1.2). These data indicated GDC-0941 does notcombine equally well with all chemotherapies. The CI's obtained withGDC-0941 and Bortezomib were generally in the range of 0.8 and higher,whereas the CI's obtained with GDC-0941 plus dexamethasone were lowerand indicated both a synergistic and more prevalent response across thecell lines tested.

TABLE 4 EC50 (μM) multiple GDC-0941 myeloma (single CI at EC50 CI atEC50 CI at EC50 CI at EC50 CI at EC50 cell line agent) Dex Dox melphalanlenalidomide bortezomib EJM 1.5 Not 0.57 Not Not 0.78 determineddetermined determined MM1.S 0.22 0.55 0.68 Not 0.92 1.18 determinedMOLP-8 0.06 1.19 0.85 0.59 0.90 Not determined NCI-H929 0.43 0.45 1.00Not 0.48 Not determined determined OPM2 1.23 0.45 0.99 2.00 0.47 0.92RPMI-8226 2.53 0.17 0.83 0.75 0.59 1.00 KSM-12- >5 Not 0.52 0.30 Not0.68 BM determined determined

TABLE 5 Combination of GDC-0941 and vincristine cell line CI at ED50 CIat ED75 CI at ED90 Bjab 0.84 0.63 0.58 DoHH2 0.92 0.94 1.03 WSU-DHL40.53 0.47 0.42 WSU-DLCL2 0.63 0.60 0.56

Table 5 shows the Combination Index scores, calculated by the method ofChou and Talalay, of different lymphoma cell lines by the therapeuticcombinations of Formula Ia compound GDC-0941 and the chemotherapeuticagent vincristine. Certain combinations show synergy (CI<0.8),additivity (0.8-1.2), or antagonism (>1.2). The data indicate thatGDC-0941 combines quite favorably with vincristine particularly in BJAB,WSU-DHL4, and WSU-DLCL2. The CI value is shown at three different pointson the dose-response curve, the ED50, ED75, and ED90 and the fact thatsimilar CI values are obtained at these different points on the doseresponse curve indicates that the data are robust and the entireresponse curve has shifted to yield an increased biological response inthe case of the combination of agents.

Table 6 shows the Combination Index scores, calculated by the Chou &Talalay method, of treatment of various hematopoietic malignancy celllines in the presence or absence of growth factors IL6 and IGF-1 by thetherapeutic combinations of Formula Ia compound (GDC-0941) anddexamethasone. Certain combinations show synergy (CI<0.8), additivity(0.8-1.2), or antagonism (>1.2). The data indicate that GDC-0941combines quite favorably with dexamethasone. The CI value is shown atthree different points on the dose-response curve, the ED50, ED75, andED90 and the fact that similar CI values are obtained at these differentpoints on the dose response curve indicates that the data are robust andthe entire response curve has shifted to yield an increased biologicalresponse in the case of the combination of agents. MM1.s cells are knownto be sensitive to dexamethasone and exhibited clear synergy whencombined with GDC-0941. The MM1.r variant of this cell line is known tobe dexamethasone resistant, and consistent with this property, overalllesser CI values were observed. The cytokines IL-6 and IGF-1 are majorgrowth factors in the bone marrow microenvironment of multiple myelomaand involved in mediating signals via the PI3K/AKT signaling pathway.The growth factors IL-6 and IGF-1 are generally thought to providechemoresistance and in each of the cell lines addition of cytokinesincreased the combination index values.

TABLE 6 Combination of GDC-0941 and dexamethasone cell line CI at ED50CI at ED75 CI at ED90 MM1.S (no IL6 or IGF1) 0.15 0.17 0.19 MM1.S (+IL6,+IGF1) 0.53 0.54 0.54 MM1.R (no IL6 or IGF1) 0.77 1.01 1.34 MM1.R (+IL6,+IGF1) 0.85 0.92 1.00 OPM2 (no IL6 or IGF1) 0.46 0.37 0.30 OPM2 (+IL6,+IGF1) 0.93 1.10 1.30 NCIH929 (no IL6 or IGF1) 0.48 0.41 0.35 NCIH929(+IL6, +IGF1) 1.08 1.39 1.80 KMS-11 (no IL6 or IGF1) 0.14 0.10 0.08KMS-11 (+IL6, +IGF1) 0.31 0.27 0.23 RPMI-8826 (no IL6 or IGF1) 0.45 0.470.49 RPMI-8826 (+IL6, +IGF1) 0.19 0.09 0.04 U266 (no IL6 or IGF1) 1.454.74 15 U266 (+IL6, +IGF1) no calc no calc no calc

Table 7 shows the Combination Index scores, calculated by the Chou &Talalay method, of treatment of various hematopoietic malignancy celllines in the presence or absence of growth factors IL6 and IGF-1 by thetherapeutic combinations of Formula Ia compound (GDC-0941) andlenalidomide. Certain combinations show synergy (CI<0.8), additivity(0.8-1.2), or antagonism (>1.2). The data indicate that GDC-0941combines quite favorably with lenalidomide. The CI value is shown atthree different points on the dose-response curve, the ED50, ED75, andED90 and for certain cell lines, the fact that similar CI values areobtained at these different points on the dose response curve indicatesthat the data are robust and the entire response curve has shifted toyield an increased biological response in the case of the combination ofagents. The growth factors IL-6 and IGF-1 are generally thought toprovide chemoresistance and in each of the cell lines addition ofcytokines increased the combination index values.

TABLE 7 Combination of GDC-0941 and lenalidomide cell line CI at ED50 CIat ED75 CI at ED90 MM1.S (no IL6 or IGF1) 0.37 0.89 2.14 MM1.S (+IL6,+IGF1) 1.01 1.06 1.11 MM1.R (no IL6 or IGF1) 0.61 0.74 0.91 MM1.R (+IL6,+IGF1) 0.85 0.92 1.00 OPM2 (no IL6 or IGF1) 0.59 0.60 0.60 OPM2 (+IL6,+IGF1) 0.70 0.78 0.86 NCIH929 (no IL6 or IGF1) 0.54 0.63 0.74 NCIH929(+IL6, +IGF1) 0.79 0.95 1.13 KMS-11 (no IL6 or IGF1) 0.53 0.72 0.96KMS-11 (+IL6, +IGF1) 0.89 0.96 1.04 RPMI-8826 (no IL6 or IGF1) 0.45 0.640.91 RPMI-8826 (+IL6, +IGF1) 1.86 2.13 2.45

Apoptotic responses to single agents FIGS. Ia and Ib compounds, and thecombinations of: (i) FIG. Ia compound (GDC-0941) and rapamycin, and (ii)FIG. Ib compound and rapamycin in multiple myeloma and AML cell lines,including OPM2 and H929, were measured by Annexin V-FACS analysis. Thescreening conditions to measure absolute IC50s comprised: Day 1—Platecells at 10,000 cells/well on a 384-well plate in media with 10% FBS;Day 2—Dose cells with indicated compound setup. When rapamycin was usedin combination with FIG. Ia or FIG. Ib compounds, rapamycinconcentration in the media was 0.1 uM; and Day 5-Celltiter-Glo assay.The measured apoptotic populations demonstrated synergy withcombinations (i) and (ii).

In blast cells from AML patients, the combination of FIG. Ia GDC-0941and a chemotherapy agent cytarabine or daunorubicin showed enhancedanti-leukemic activity compared to single agent GDC-0941, and enhancedapoptic efficacy with only 30% and 22% live cells remaining,respectively.

In Vivo Tumor Xenograft Efficacy

The efficacy of the combinations of the invention may be measured invivo by implanting allografts or xenografts of cancer cells in rodentsand treating the tumor-bearing animals with the combinations. Variableresults are to be expected depending on the cell line, the presence orabsence of certain mutations in the tumor cells, the sequence ofadministration of Formula I compound and chemotherapeutic agent, dosingregimen, and other factors. Subject mice were treated with drug(s) orcontrol (Vehicle) and monitored over several weeks or more to measurethe time to tumor doubling, log cell kill, and tumor inhibition (Example16).

FIG. 7 shows the mean tumor volume change over 20 days in cohorts of 10mice with WSU-DLCL2 lymphoma tumor xenografts dosed on day 0 withVehicle (0.5% Methylcellulose: 0.2% Tween 80 in DI Water), 73 mg/kgFormula Ia (GDC-0941), 5 mg/kg rituximab, CHOP, and the combinations ofFormula Ia 73 mg/kg and rituximab 5 mg/kg, Formula Ia 73 mg/kg and CHOP.Mice were dosed with CHOP starting on day 0, and rituximab on days 0, 7,and 14, while Formula Ia was dosed daily for 21 days by oral gavage.CHOP regimen: cyclophosphamide (30 mg/kg, iv, qd×1), doxorubicin (2.475mg/kg, iv, qd×1), vincristine (0.375 mg/kg, iv, qd×1), prednisone (0.15mg/kg, po, qd×5). Cyclophosphamide, doxorubicin and vincristine weredosed once on day 0 and prednisone was dosed on days 0, 1, 2, 3 and 4.In this model, GDC-0941, and CHOP-based combination chemotherapy hadonly modest activity. Rituximab treatment was significantly moredifferent from vehicle (p<0.01 by Control Dunnett's t-test). Thecombination of GDC-0941 with rituximab was significantly better thanGDC-0941, but not different from rituximab alone by log-rank analysis.The combination of GDC-0941 and CHOP gave a significant improvement inefficacy compared to either agent alone.

FIG. 8 shows the mean tumor volume change over 34 days in cohorts of 10mice with DoHH-2 tumor xenografts dosed on day 0 with: Vehicle (0.5%methylcellulose: 0.2% Tween 80 in DI Water), 73 mg/kg Formula Ia(GDC-0941), 5 mg/kg rituximab, CHOP, and the combinations of Formula Ia73 mg/kg and rituximab 5 mg/kg, and Formula Ia 100 mg/kg and CHOP. Micewere dosed with rituximab on day 0, 7 and 14 (qwk×3) intravenously, CHOPstarting on day 1, while Formula Ia was dosed daily for 21 days by oralgavage. CHOP regimen: cyclophosphamide (30 mg/kg, iv, qd×1), doxorubicin(2.475 mg/kg, iv, qd×1), vincristine (0.375 mg/kg, iv, qd×1), prednisone(0.15 mg/kg, po, qd×5). Cyclophosphamide, doxorubicin and vincristinewere dosed once on day 0 and prednisone was dosed on days 0, 1, 2, 3 and4. CHOP chemotherapy or GDC-0941 monotherapy cohorts were significantlydifferent than vehicle. Rituximab monotherapy was relatively moreeffective causing 2 partial responses and 4 complete responses duringtreatment. GDC-0941 did not significantly antagonize rituximab activity,however, it did not provide additional antitumor activity. In contrast,the combination of GDC-0941 with CHOP chemotherapy gave a verysignificant increase in benefit over the activity of either single agentand produced 2 partial responses and 2 complete responses.

FIG. 9 shows the mean tumor volume change over 27 days in cohorts of 10mice with DoHH2 tumor xenografts dosed on day 0 with: Vehicle (0.5%methylcellulose: 0.2% Tween 80 in DI Water), 75 mg/kg Formula Ia(GDC-0941), CHOP, and the combinations of Formula Ia 75 mg/kg and CHOP,Formula Ia 75 mg/kg and cyclophosphamide 30 mg/kg, Formula Ia 75 mg/kgand doxorubicin 2.47 mg/kg, Formula Ia 75 mg/kg and vincristine 0.38mg/kg, and Formula Ia 75 mg/kg and prednisone 0.15 mg/kg. Mice weredosed with CHOP on day 0, cyclophosphamide on day 0, doxorubicin on day0, vincristine on day 0, and prednisone daily on days 0-4, while FormulaIa was dosed daily for 21 days by oral gavage. CHOP components regimenwas: cyclophosphamide (30 mg/kg, iv, qd×1), doxorubicin (2.475 mg/kg,iv, qd×1), vincristine (0.375 mg/kg, iv, qd×1), prednisone (0.15 mg/kg,po, qd×5). This experiment confirms and extends the results of FIG. 8 toshow that GDC-0941 and CHOP each have similar and only moderate activityin the model. As before, GDC-0941 combines with CHOP to give a verysignificant increase in anti-tumor activity. Surprisingly, as this wasnot predicted from in vitro experiments, essentially all of the synergynoted between GDC-0941 and CHOP can be attributed to the combination ofjust GDC-0941 and vincristine, whereas the other three components ofCHOP tested pairwise with GDC-0941 did not exhibit increased efficacy.

FIG. 10 shows the mean tumor volume change over 25 days in cohorts of 10mice with BJAB lymphoma tumor xenografts dosed on day 0 with: Vehicle(0.5% Methylcellulose: 0.2% Tween 80 in DI Water), 73 mg/kg Formula Ia(GDC-0941), CHOP, and the combination of Formula Ia 73 mg/kg and CHOP.Mice were dosed once with CHOP starting on day 0, while Formula Ia andVehicle were dosed daily for 21 days by oral gavage. CHOP regimen:cyclophosphamide (30 mg/kg, iv, qd×1), doxorubicin (2.475 mg/kg, iv,qd×1), vincristine (0.375 mg/kg, iv, qd×1), prednisone (0.15 mg/kg, po,qd×5). Cyclophosphamide, doxorubicin and vincristine were dosed once onday 0 and prednisone was dosed on days 0, 1, 2, 3 and 4. These data showin the BJAB lymphoma model that GDC-0941 or CHOP chemotherapy have onlymodest activity and that the combination has increased activity althoughno groups reach statistical significance.

FIG. 11 shows the mean tumor volume change over 25 days in cohorts of 10mice with BJAB lymphoma tumor xenografts dosed on day 0 with: Vehicle(0.5% Methylcellulose: 0.2% Tween 80 in DI Water), 75 mg/kg Formula Ia(GDC-0941), 5 mg/kg rituximab, and the combination of Formula Ia 75mg/kg and 5 mg/kg rituximab. Mice were dosed with rituximab on days 0,7, and 14, while Formula Ia and Vehicle were dosed daily for 21 days(po, qd×21) by oral gavage. As in FIG. 10, single agent GDC-0941 hadonly modest activity in this BJAB lymphoma model. The activity ofrituximab was modest compared to single agent GDC-0941 and was notfurther increased by combination with GDC-0941. In this study all groupsare significantly different than vehicle by log-rank analysis.

FIG. 12 shows the mean tumor volume change over 22 days in cohorts of 10mice with NCI-H929 multiple myeloma xenografts dosed on day 0 with:Vehicle (po, qd×21) (0.5% Methylcellulose: 0.2% Tween 80 in DI Water),and single agent therapies: 73 mg/kg Formula Ia GDC-0941 (po, qd×21), 1mg/kg bortezomib (iv, 2×/wk×3), 25 mg/kg lenalidomide (ip, qd×21), and10 mg/kg dexamethasone (po, 5 days on/2 days off/4 days on). Formula IaGDC-0941 was dosed daily for 21 days by oral gavage. Bortezomib wasdosed intravenously on days 0, 3, 7, 10, 14 and 17. Lenalidomide wasdosed on daily for 21 days by intraperitoneal injection. Dexamethasonewas dosed orally on days 0-4 and 7-10. This experiment established thesingle agent activity of GDC-0941 as similar to that of lenalidomide ordexamethasone single agent treatments, which were exceeded by theefficacy of bortezomib. Bortezomib single agent treatment resulted in 3partial responses in this study.

FIG. 13 shows the mean tumor volume change over 24 days in cohorts of 10mice with multiple myeloma OPM-2 cell xenografts dosed on day 0 with:Vehicle (po, qd×21) (0.5% Methylcellulose: 0.2% Tween 80 in DI Water),single agent therapies: 73 mg/kg Formula Ia GDC-0941 (po, qd×21); 0.5mg/kg bortezomib (iv, 2×/wk×3); 25 mg/kg lenalidomide (ip, 5 days on/2days off/5 days on/2 days off/5 days on); and 3 mg/kg dexamethasone (po,5 days on/2 days off/5 days on); and combinations of: 73 mg/kg FormulaIa GDC-0941 (po, qd×21) and 0.5 mg/kg bortezomib (iv, 2×/wk×3); 73 mg/kgFormula Ia GDC-0941 (po, qd×21) and 25 mg/kg lenalidomide (ip,5/2/5/2/5); and 73 mg/kg Formula Ia GDC-0941 (po, qd×21) and 3 mg/kgdexamethasone (po, 5/2/5). Formula Ia GDC-0941 was dosed daily for 21days by oral gavage. Bortezomib was dosed intravenously on days 0, 3, 7,10, 14 and 17. Lenalidomide was dosed on days 0-4, 7-11 and 14-18 byintraperitoneal injection. Dexamethasone was dosed orally on days 0-4and 7-11. A reduced dose of bortezomib in this experiment resulted in asub-clinical response which was not different than that of vehicle.Cohorts treated with single agent GDC-0941, lenalidomide, ordexamethasone had an indistinguishable and moderate tumor activity,though the addition of GDC-0941 to dexamethasone trended towardsincreased antitumor activity. This result was predicted from thepreceding in vitro cell line studies but not predictable from priorstudies published in the literature.

FIG. 14 shows the mean tumor volume change over 27 days in cohorts of 10mice with multiple myeloma MM1.s cell xenografts dosed on day 0 with:Vehicle (po, qd×21) (0.5% Methylcellulose: 0.2% Tween 80 in DI Water),73 mg/kg Formula Ia GDC-0941 (po, qd×21), 1 mg/kg bortezomib (iv,2×/wk×2.5), 25 mg/kg lenalidomide (ip, qd×21), and 10 mg/kgdexamethasone (po, 5 days on/2 days off/5 days on). Formula Ia GDC-0941was dosed daily for 21 days by oral gavage. Bortezomib was dosedintravenously on days 0, 3, 7, and 14. Lenalidomide was dosed on dailyfor 21 days by intraperitoneal injection. Dexamethasone was dosed orallyon days 0-4 and 7-11. This experiment established the single agentactivity of GDC-0941 as similar to that of lenalidomide, bortezomib, ordexamethasone single agent treatments.

FIG. 15 shows the mean tumor volume change over 40 days in cohorts of 10mice with multiple myeloma MM1.s cell xenografts dosed on day 0 with:Vehicle (po, qd×21) (0.5% Methylcellulose: 0.2% Tween 80 in DI Water),single agent therapies: 75 mg/kg Formula Ia GDC-0941 (po, qd×21), 0.5mg/kg bortezomib (iv, 2×/wk×3), and 3 mg/kg dexamethasone (po, 5/2/5);and combinations of: 75 mg/kg Formula Ia GDC-0941 (po, qd×21) and 0.5mg/kg bortezomib (iv, 2×/wk×3); and 75 mg/kg Formula Ia GDC-0941 (po,qd×21) and 3 mg/kg dexamethasone (po, 5/2/5). Formula Ia GDC-0941 wasdosed daily for 21 days by oral gavage. Bortezomib was dosedintravenously on days 0, 3, 7, 10, 14 and 17. Dexamethasone was dosedorally on days 0-4 and 7-11. The MM1.s model was relatively refractoryto these single agent and combination treatments with one exception;consistent with in vitro experiments, the GDC-0941 and dexamethasonecombination had excellent activity compared to the single agentcomponents. Surprising and unexpectedly, during the time that theGDC-0941 plus dexamethasone cohort were on combination drug treatment,the tumors regressed producing 7 partial responses. No other groups inthis study produced objective responses. When dexamethasone treatmentwas discontinued on day 11, tumors ceased to regress and grew at a rateconsistent with continued GDC-0941. These data clearly indicate theunexpected efficacy of the combination of dexamethasone and GDC-0941.

FIG. 16 shows the mean tumor volume change over 33 days in cohorts of 10mice with multiple myeloma NCI-H929 cell xenografts dosed on day 0 with:Vehicle (po, qd×21) (0.5% Methylcellulose: 0.2% Tween 80 in DI Water)single agent therapies: 75 mg/kg Formula Ia GDC-0941 (po, qd×21), 0.5mg/kg bortezomib (iv, 2×/wk×3), 25 mg/kg lenalidomide (ip, 5/2/5/2/5),and 3 mg/kg dexamethasone (po, 5/2/5); and combinations of: 75 mg/kgFormula Ia GDC-0941 (po, qd×21) and 0.5 mg/kg bortezomib (iv, qd×3); 75mg/kg Formula Ia GDC-0941 (po, qd×21) and 25 mg/kg lenalidomide (ip,5/2/5/2/5); and 75 mg/kg Formula Ia GDC-0941 (po, qd×14) and 3 mg/kgdexamethasone (po, 5/2/5). Formula Ia GDC-0941 was dosed daily for 21days by oral gavage. Bortezomib was dosed intravenously on days 0, 3, 7,10, 14 and 17. Lenalidomide was dosed on days 0-4, 7-11 and 14-18 byintraperitoneal injection. Dexamethasone was dosed orally on days 0-4and 7-11. In the H929 model, the addition of GDC-0941 in the combinationgroups significantly increased the modest activity of single agentbortezomib, lenalidomide, and dexamethasone.

FIG. 17 shows the mean tumor volume change over 40 days for experimentalcohorts of 10 mice per group bearing pre-established DoHH2 humanlymphoma cell line xenografts dosed on day 0 with: Vehicle (po, qd×20))0.5% Methylcellulose: 0.2% Tween 80 in DI Water); single agenttherapies: 75 mg/kg or 100 mg/kg Formula Ia GDC-0941 (po, qd×20), 2.5mg/kg or 4 mg/kg Formula Ib (po, qd×20), 6 mg/kg Rapamycin (ip, qw×3);or combination therapies: 75 mg/kg Formula Ia GDC-0941 (po, qd×20) plus6 mg/kg Rapamycin (ip, qw×3), or 2.5 mg/kg Formula Ib (po, qd×20) plus 6mg/kg Rapamycin (ip, qw×3). Rapamycin shows little or no significanteffect on tumor growth, whereas the single agent treatments showdose-related inhibition of tumor growth while both combinations showsignificantly enhanced suppression of tumor growth.

FIG. 18 shows the mean tumor volume change over 25 days for experimentalcohorts of 10 mice per group bearing pre-established WSU-DLCL2 humanlymphoma cell line xenografts dosed on day 0 with: Vehicle (po, qd×20))0.5% Methylcellulose: 0.2% Tween 80 in DI Water); single agenttherapies: 60 mg/kg Formula Ia GDC-0941 (po, qd×21), 1 mg/kg Formula Ib(po, qd×21), 6 mg/kg Rapamycin (ip, qd×21); or combination therapies: 60mg/kg Formula Ia GDC-0941 (po, qd×18) plus 6 mg/kg Rapamycin (ip,qd×18), or 1 mg/kg Formula Ib (po, qd×18) plus 6 mg/kg Rapamycin (ip,qd×18). The single agent treatments show little effect on tumor growth,whereas combinations show significantly enhanced suppression of tumorgrowth.

Pharmaceutical Compositions

Pharmaceutical compositions or formulations of the present inventioninclude combinations of Formula I compounds, a chemotherapeutic agent,and one or more pharmaceutically acceptable carrier, glidant, diluent,or excipient.

The Formula I compounds, and chemotherapeutic agents of the presentinvention may exist in unsolvated as well as solvated forms withpharmaceutically acceptable solvents such as water, ethanol, and thelike, and it is intended that the invention embrace both solvated andunsolvated forms.

The Formula I compounds, and chemotherapeutic agents of the presentinvention may also exist in different tautomeric forms, and all suchforms are embraced within the scope of the invention. The term“tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

Pharmaceutical compositions encompass both the bulk composition andindividual dosage units comprised of more than one (e.g., two)pharmaceutically active agents including a Formula I compound and achemotherapeutic agent selected from the lists of the additional agentsdescribed herein, along with any pharmaceutically inactive excipients,diluents, carriers, or glidants. The bulk composition and eachindividual dosage unit can contain fixed amounts of the aforesaidpharmaceutically active agents. The bulk composition is material thathas not yet been formed into individual dosage units. An illustrativedosage unit is an oral dosage unit such as tablets, pills, capsules, andthe like. Similarly, the methods of treating a patient by administeringa pharmaceutical composition is also intended to encompass theadministration of the bulk composition and individual dosage units.

Pharmaceutical compositions also embrace isotopically-labeled compoundsof the present invention which are identical to those recited herein,but for the fact that one or more atoms are replaced by an atom havingan atomic mass or mass number different from the atomic mass or massnumber usually found in nature. All isotopes of any particular atom orelement as specified are contemplated within the scope of the compoundsof the invention, and their uses. Exemplary isotopes that can beincorporated into compounds of the invention include isotopes ofhydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine,chlorine and iodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O,¹⁸O, ³²P, ³³P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I and ¹²⁵I. Certainisotopically-labeled compounds of the present invention (e.g., thoselabeled with ³H and ¹⁴C) are useful in compound and/or substrate tissuedistribution assays. Tritiated (³H) and carbon-14 (¹⁴C) isotopes areuseful for their ease of preparation and detectability. Further,substitution with heavier isotopes such as deuterium (²H) may affordcertain therapeutic advantages resulting from greater metabolicstability (e.g., increased in vivo half-life or reduced dosagerequirements) and hence may be preferred in some circumstances. Positronemitting isotopes such as ¹⁵O, ¹³N, ¹¹C and ¹⁸F are useful for positronemission tomography (PET) studies to examine substrate receptoroccupancy. Isotopically labeled compounds of the present invention cangenerally be prepared by following procedures analogous to thosedisclosed in the Schemes and/or in the Examples herein below, bysubstituting an isotopically labeled reagent for a non-isotopicallylabeled reagent.

Formula I compounds and chemotherapeutic agents are formulated inaccordance with standard pharmaceutical practice for use in atherapeutic combination for therapeutic treatment (includingprophylactic treatment) of hyperproliferative disorders in mammalsincluding humans. The invention provides a pharmaceutical compositioncomprising a Formula I compound in association with one or morepharmaceutically acceptable carrier, glidant, diluent, additive, orexcipient.

Suitable carriers, diluents, additives, and excipients are well known tothose skilled in the art and include materials such as carbohydrates,waxes, water soluble and/or swellable polymers, hydrophilic orhydrophobic materials, gelatin, oils, solvents, water and the like. Theparticular carrier, diluent or excipient used will depend upon the meansand purpose for which the compound of the present invention is beingapplied. Solvents are generally selected based on solvents recognized bypersons skilled in the art as safe (GRAS) to be administered to amammal. In general, safe solvents are non-toxic aqueous solvents such aswater and other non-toxic solvents that are soluble or miscible inwater. Suitable aqueous solvents include water, ethanol, propyleneglycol, polyethylene glycols (e.g., PEG 400, PEG 300), dimethylsulfoxide(DMSO), cremophor (e.g. CREMOPHOR EL®, BASF). and mixtures thereof. Theformulations may also include one or more buffers, stabilizing agents,surfactants, wetting agents, lubricating agents, emulsifiers, suspendingagents, preservatives, antioxidants, opaquing agents, glidants,processing aids, colorants, sweeteners, perfuming agents, flavoringagents and other known additives to provide an elegant presentation ofthe drug (i.e., a compound of the present invention or pharmaceuticalcomposition thereof) or aid in the manufacturing of the pharmaceuticalproduct (i.e., medicament).

The formulations may be prepared using conventional dissolution andmixing procedures. For example, the bulk drug substance (i.e., compoundof the present invention or stabilized form of the compound (e.g.,complex with a cyclodextrin derivative or other known complexationagent) is dissolved in a suitable solvent in the presence of one or moreof the excipients described above. The compound of the present inventionis typically formulated into pharmaceutical dosage forms to provide aneasily controllable dosage of the drug and to enable patient compliancewith the prescribed regimen.

The pharmaceutical composition (or formulation) for application may bepackaged in a variety of ways depending upon the method used foradministering the drug. Generally, an article for distribution includesa container having deposited therein the pharmaceutical formulation inan appropriate form. Suitable containers are well known to those skilledin the art and include materials such as bottles (plastic and glass),sachets, ampoules, plastic bags, metal cylinders, and the like. Thecontainer may also include a tamper-proof assemblage to preventindiscreet access to the contents of the package. In addition, thecontainer has deposited thereon a label that describes the contents ofthe container. The label may also include appropriate warnings.

Pharmaceutical formulations of the compounds of the present inventionmay be prepared for various routes and types of administration. Forexample, a Formula I compound having the desired degree of purity mayoptionally be mixed with pharmaceutically acceptable diluents, carriers,excipients or stabilizers (Remington's Pharmaceutical Sciences (1995)18th edition, Mack Publ. Co., Easton, Pa.), in the form of a lyophilizedformulation, milled powder, or an aqueous solution. Formulation may beconducted by mixing at ambient temperature at the appropriate pH, and atthe desired degree of purity, with physiologically acceptable carriers,i.e., carriers that are non-toxic to recipients at the dosages andconcentrations employed. The pH of the formulation depends mainly on theparticular use and the concentration of compound, but may range fromabout 3 to about 8.

The pharmaceutical formulation is preferably sterile. In particular,formulations to be used for in vivo administration must be sterile. Suchsterilization is readily accomplished by filtration through sterilefiltration membranes.

The pharmaceutical formulation ordinarily can be stored as a solidcomposition, a lyophilized formulation or as an aqueous solution.

The pharmaceutical formulations of the invention will be dosed andadministered in a fashion, i.e., amounts, concentrations, schedules,course, vehicles and route of administration, consistent with goodmedical practice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the compound to be administered will be governed by suchconsiderations, and is the minimum amount necessary to prevent,ameliorate, or treat the coagulation factor mediated disorder. Suchamount is preferably below the amount that is toxic to the host orrenders the host significantly more susceptible to bleeding.

The initial pharmaceutically effective amount of the Formula I compoundadministered orally or parenterally per dose will be in the range ofabout 0.01-1000 mg/kg, namely about 0.1 to 20 mg/kg of patient bodyweight per day, with the typical initial range of compound used being0.3 to 15 mg/kg/day. The dose of the Formula I compound and the dose ofthe chemotherapeutic agent to be administered may range for each fromabout 1 mg to about 1000 mg per unit dosage form, or from about 10 mg toabout 100 mg per unit dosage form. The doses of Formula I compound andthe chemotherapeutic agent may administered in a ratio of about 1:50 toabout 50:1 by weight, or in a ratio of about 1:10 to about 10:1 byweight.

Acceptable diluents, carriers, excipients and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, CREMOPHOR EL®, PLURONICS™ or polyethyleneglycol (PEG). The active pharmaceutical ingredients may also beentrapped in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences18th edition, (1995) Mack Publ. Co., Easton, Pa.

Sustained-release preparations of Formula I compounds may be prepared.Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing acompound of Formula I, which matrices are in the form of shapedarticles, e.g., films, or microcapsules. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate) and poly-D(−)3-hydroxybutyric acid.

The pharmaceutical formulations include those suitable for theadministration routes detailed herein. The formulations may convenientlybe presented in unit dosage form and may be prepared by any of themethods well known in the art of pharmacy. Techniques and formulationsgenerally are found in Remington's Pharmaceutical Sciences 18^(th) Ed.(1995) Mack Publishing Co., Easton, Pa. Such methods include the step ofbringing into association the active ingredient with the carrier whichconstitutes one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both, and then, if necessary, shaping the product.

Formulations of a compound of Formula I and/or chemotherapeutic agentsuitable for oral administration may be prepared as discrete units suchas pills, hard or soft e.g., gelatin capsules, cachets, troches,lozenges, aqueous or oil suspensions, dispersible powders or granules,emulsions, syrups or elixirs each containing a predetermined amount of acompound of Formula I and/or a chemotherapeutic agent. The amount ofcompound of Formula I and the amount of chemotherapeutic agent may beformulated in a pill, capsule, solution or suspension as a combinedformulation. Alternatively, the Formula I compound and thechemotherapeutic agent may be formulated separately in a pill, capsule,solution or suspension for administration by alternation.

Formulations may be prepared according to any method known to the artfor the manufacture of pharmaceutical compositions and such compositionsmay contain one or more agents including sweetening agents, flavoringagents, coloring agents and preserving agents, in order to provide apalatable preparation. Compressed tablets may be prepared by compressingin a suitable machine the active ingredient in a free-flowing form suchas a powder or granules, optionally mixed with a binder, lubricant,inert diluent, preservative, surface active or dispersing agent. Moldedtablets may be made by molding in a suitable machine a mixture of thepowdered active ingredient moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and optionally are formulatedso as to provide slow or controlled release of the active ingredienttherefrom.

Tablet excipients of a pharmaceutical formulation of the invention mayinclude: Filler (or diluent) to increase the bulk volume of the powdereddrug making up the tablet; Disintegrants to encourage the tablet tobreak down into small fragments, ideally individual drug particles, whenit is ingested and promote the rapid dissolution and absorption of drug;Binder to ensure that granules and tablets can be formed with therequired mechanical strength and hold a tablet together after it hasbeen compressed, preventing it from breaking down into its componentpowders during packaging, shipping and routine handling; Glidant toimprove the flowability of the powder making up the tablet duringproduction; Lubricant to ensure that the tabletting powder does notadhere to the equipment used to press the tablet during manufacture.They improve the flow of the powder mixes through the presses andminimize friction and breakage as the finished tablets are ejected fromthe equipment; Antiadherent with function similar to that of theglidant, reducing adhesion between the powder making up the tablet andthe machine that is used to punch out the shape of the tablet duringmanufacture; Flavor incorporated into tablets to give them a morepleasant taste or to mask an unpleasant one, and Colorant to aididentification and patient compliance.

Tablets containing the active ingredient in admixture with non-toxicpharmaceutically acceptable excipient which are suitable for manufactureof tablets are acceptable. These excipients may be, for example, inertdiluents, such as calcium or sodium carbonate, lactose, calcium orsodium phosphate; granulating and disintegrating agents, such as maizestarch, or alginic acid; binding agents, such as starch, gelatin oracacia; and lubricating agents, such as magnesium stearate, stearic acidor talc. Tablets may be uncoated or may be coated by known techniquesincluding microencapsulation to delay disintegration and adsorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

For treatment of the eye or other external tissues, e.g., mouth andskin, the formulations are preferably applied as a topical ointment orcream containing the active ingredient(s) in an amount of, for example,0.075 to 20% w/w. When formulated in an ointment, the active ingredientsmay be employed with either a paraffinic or a water-miscible ointmentbase. Alternatively, the active ingredients may be formulated in a creamwith an oil-in-water cream base.

The aqueous phase of the cream base may include a polyhydric alcohol,i.e., an alcohol having two or more hydroxyl groups such as propyleneglycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethyleneglycol (including PEG 400) and mixtures thereof. The topicalformulations may desirably include a compound which enhances absorptionor penetration of the active ingredient through the skin or otheraffected areas. Examples of such dermal penetration enhancers includedimethyl sulfoxide and related analogs.

The oily phase of the emulsions of this invention may be constitutedfrom known ingredients in a known manner, including a mixture of atleast one emulsifier with a fat or an oil, or with both a fat and anoil. Preferably, a hydrophilic emulsifier is included together with alipophilic emulsifier which acts as a stabilizer. Together, theemulsifier(s) with or without stabilizer(s) make up an emulsifying wax,and the wax together with the oil and fat comprise an emulsifyingointment base which forms the oily dispersed phase of creamformulations. Emulsifiers and emulsion stabilizers suitable for use inthe formulation of the invention include Tween® 60, Span® 80,cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glycerylmono-stearate and sodium lauryl sulfate.

Aqueous suspensions of the pharmaceutical formulations of the inventioncontain the active materials in admixture with excipients suitable forthe manufacture of aqueous suspensions. Such excipients include asuspending agent, such as sodium carboxymethylcellulose, croscarmellose,povidone, methylcellulose, hydroxypropyl methylcellulose, sodiumalginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, anddispersing or wetting agents such as a naturally occurring phosphatide(e.g., lecithin), a condensation product of an alkylene oxide with afatty acid (e.g., polyoxyethylene stearate), a condensation product ofethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxybenzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.

Pharmaceutical compositions may be in the form of a sterile injectablepreparation, such as a sterile injectable aqueous or oleaginoussuspension. This suspension may be formulated according to the known artusing those suitable dispersing or wetting agents and suspending agentswhich have been mentioned above. The sterile injectable preparation maybe a solution or a suspension in a non-toxic parenterally acceptablediluent or solvent, such as a solution in 1,3-butanediol or preparedfrom a lyophilized powder. Among the acceptable vehicles and solventsthat may be employed are water, Ringer's solution and isotonic sodiumchloride solution. In addition, sterile fixed oils may conventionally beemployed as a solvent or suspending medium. For this purpose any blandfixed oil may be employed including synthetic mono- or diglycerides. Inaddition, fatty acids such as oleic acid may likewise be used in thepreparation of injectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Formulations suitable for topical administration to the eye also includeeye drops wherein the active ingredient is dissolved or suspended in asuitable carrier, especially an aqueous solvent for the activeingredient. The active ingredient is preferably present in suchformulations in a concentration of about 0.5 to 20% w/w, for exampleabout 0.5 to 10% w/w, for example about 1.5% w/w.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavored basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouthwashes comprising the active ingredient in asuitable liquid carrier.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising for example cocoa butter or asalicylate.

Formulations suitable for intrapulmonary or nasal administration have aparticle size for example in the range of 0.1 to 500 microns (includingparticle sizes in a range between 0.1 and 500 microns in incrementsmicrons such as 0.5, 1, 30 microns, 35 microns, etc.), which isadministered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis disorders as described below.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such carriers as areknown in the art to be appropriate.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefore. Veterinary carriers are materials useful for thepurpose of administering the composition and may be solid, liquid orgaseous materials which are otherwise inert or acceptable in theveterinary art and are compatible with the active ingredient. Theseveterinary compositions may be administered parenterally, orally or byany other desired route.

Combination Therapy

Formula I compounds may be employed in combination with certainchemotherapeutic agents for the treatment of a hematopoietic malignancy,along with pre-malignant and non-neoplastic or non-malignanthyperproliferative disorders. In certain embodiments, a compound ofFormula I is combined in a pharmaceutical combination formulation, ordosing regimen as combination therapy, with a chemotherapeutic agentthat has anti-hyperproliferative properties or that is useful fortreating the hematopoietic malignancy. The chemotherapeutic agent of thepharmaceutical combination formulation or dosing regimen preferably hascomplementary activities to the Formula I compound, and such that theydo not adversely affect each other. Such compounds of the therapeuticcombination may be administered in amounts that are effective for thepurpose intended. In one embodiment, a pharmaceutical formulation ofthis invention comprises a Formula I compound and a chemotherapeuticagent such as described herein. In another embodiment, the therapeuticcombination is administered by a dosing regimen wherein thetherapeutically effective amount of a Formula I compound is administeredin a range from twice daily to once every three weeks (q3wk), and thetherapeutically effective amount of the chemotherapeutic agent isadministered separately, in alternation, in a range from twice daily toonce every three weeks.

Therapeutic combinations of the invention include a product comprising aFormula I compound, and a chemotherapeutic agent selected fromdexamethasone, thioTEPA, doxorubicin, vincristine, rituximab,cyclophosphamide, prednisone, melphalan, lenalidomide, bortezomib,rapamycin, and cytarabine, as a combined preparation for separate,simultaneous or sequential use in the treatment of a hyperproliferativedisorder.

The combination therapy may be administered as a simultaneous orsequential regimen. When administered sequentially, the combination maybe administered in two or more administrations. The combinedadministration includes coadministration, using separate formulations ora single pharmaceutical formulation, and consecutive administration ineither order, wherein preferably there is a time period while both (orall) active agents simultaneously exert their biological activities.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other chemotherapeutic agents ortreatments, such as to increase the therapeutic index or mitigatetoxicity or other side-effects or consequences.

In a particular embodiment of anti-cancer therapy, the therapeuticcombination may be combined with surgical therapy and radiotherapy, asadjuvant therapy. Combination therapies according to the presentinvention include the administration of at least one Formula I compoundand one or more other cancer treatment methods or modalities. Theamounts of the Formula I compound(s) and the chemotherapeutic agent(s)and the relative timings of administration will be selected in order toachieve the desired combined therapeutic effect.

Administration of Pharmaceutical Compositions

The therapeutic combinations of the invention may be administered by anyroute appropriate to the condition to be treated. Suitable routesinclude oral, parenteral (including subcutaneous, intramuscular,intravenous, intraarterial, inhalation, intradermal, intrathecal,epidural, and infusion techniques), transdermal, rectal, nasal, topical(including buccal and sublingual), vaginal, intraperitoneal,intrapulmonary and intranasal. Topical administration can also involvethe use of transdermal administration such as transdermal patches oriontophoresis devices. Formulation of drugs is discussed in Remington'sPharmaceutical Sciences, 18^(th) Ed., (1995) Mack Publishing Co.,Easton, Pa. Other examples of drug formulations can be found inLiberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,Marcel Decker, Vol 3, 2^(nd) Ed., New York, N.Y. For localimmunosuppressive treatment, the compounds may be administered byintralesional administration, including perfusing or otherwisecontacting the graft with the inhibitor before transplantation. It willbe appreciated that the preferred route may vary with for example thecondition of the recipient. Where the compound is administered orally,it may be formulated as a pill, capsule, tablet, etc. with apharmaceutically acceptable carrier, glidant, or excipient. Where thecompound is administered parenterally, it may be formulated with apharmaceutically acceptable parenteral vehicle or diluent, and in a unitdosage injectable form, as detailed below.

A dose to treat human patients may range from about 10 mg to about 1000mg of Formula I compound, such as about 100 mg to about 300 mg of thecompound. A dose may be administered once a day (QD), twice per day(BID), or more frequently, depending on the pharmacokinetic (PK) andpharmacodynamic (PD) properties, including absorption, distribution,metabolism, and excretion of the particular compound. In addition,toxicity factors may influence the dosage and administration dosingregimen. When administered orally, the pill, capsule, or tablet may beingested twice daily, daily or less frequently such as weekly or onceevery two or three weeks for a specified period of time. The regimen maybe repeated for a number of cycles of therapy.

Methods of Treatment

Therapeutic combinations of: (1) a Formula I compound and (2) achemotherapeutic agent are useful for treating diseases, conditionsand/or disorders including, but not limited to, those characterized byactivation of the PI3 kinase pathway. Accordingly, another aspect ofthis invention includes methods of treating diseases or conditions thatcan be treated by inhibiting lipid kinases, including PI3. In oneembodiment, a method for the treatment of a hematopoietic malignancycomprises administering a therapeutic combination as a combinedformulation or by alternation to a mammal, wherein the therapeuticcombination comprises a therapeutically effective amount of a compoundhaving Formula I, and a therapeutically effective amount of one or morechemotherapeutic agents selected from dexamethasone, thioTEPA,doxorubicin, vincristine, rituximab, cyclophosphamide, prednisone,melphalan, lenalidomide, bortezomib, rapamycin, and cytarabine.Therapeutic combinations of: (1) a Formula I or II compound and (2) achemotherapeutic agent may be employed for the treatment of ahyperproliferative disease or disorder, including tumors, cancers, andneoplastic tissue, along with pre-malignant and non-neoplastic ornon-malignant hyperproliferative disorders. In one embodiment, a humanpatient is treated with a therapeutic combination and a pharmaceuticallyacceptable carrier, adjuvant, or vehicle, wherein the Formula Icompound, or metabolite thereof, of said therapeutic combination ispresent in an amount to detectably inhibit PI3 kinase activity.

Hematopoietic malignancies include non-Hodgkin's lymphoma, diffuse largehematopoietic lymphoma, follicular lymphoma, mantle cell lymphoma,chronic lymphocytic leukemia, multiple myeloma, AML, MCL

Another aspect of this invention provides a pharmaceutical compositionor therapeutic combination for use in the treatment of the diseases orconditions described herein in a mammal, for example, a human, sufferingfrom such disease or condition. Also provided is the use of apharmaceutical composition in the preparation of a medicament for thetreatment of the diseases and conditions described herein in awarm-blooded animal, such as a mammal, for example a human, sufferingfrom such disorder.

Metabolites of Formula I Compounds

Also falling within the scope of this invention are the in vivometabolic products of Formula I compounds described herein. Suchproducts may result for example from the oxidation, reduction,hydrolysis, amidation, deamidation, esterification, deesterification,enzymatic cleavage, and the like, of the administered compound.Accordingly, the invention includes metabolites of Formula I compounds,including compounds produced by a process comprising contacting acompound of this invention with a mammal for a period of time sufficientto yield a metabolic product thereof.

Metabolite products typically are identified by preparing aradiolabelled (e.g., ¹⁴C or ³H) isotope of a compound of the invention,administering it parenterally in a detectable dose (e.g., greater thanabout 0.5 mg/kg) to an animal such as rat, mouse, guinea pig, monkey, orto man, allowing sufficient time for metabolism to occur (typicallyabout 30 seconds to 30 hours) and isolating its conversion products fromthe urine, blood or other biological samples. These products are easilyisolated since they are labeled (others are isolated by the use ofantibodies capable of binding epitopes surviving in the metabolite). Themetabolite structures are determined in conventional fashion, e.g., byMS, LC/MS or NMR analysis. In general, analysis of metabolites is donein the same way as conventional drug metabolism studies well known tothose skilled in the art. The metabolite products, so long as they arenot otherwise found in vivo, are useful in diagnostic assays fortherapeutic dosing of the compounds of the invention.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing Formula I compounds useful for the treatment of thediseases and disorders described above is provided. In one embodiment,the kit comprises a container comprising a Formula I compound. The kitmay further comprise a label or package insert, on or associated withthe container. The term “package insert” is used to refer toinstructions customarily included in commercial packages of therapeuticproducts, that contain information about the indications, usage, dosage,administration, contraindications and/or warnings concerning the use ofsuch therapeutic products. Suitable containers include, for example,bottles, vials, syringes, blister pack, etc. The container may be formedfrom a variety of materials such as glass or plastic. The container mayhold a compound of Formula I or a formulation thereof which is effectivefor treating the condition and may have a sterile access port (forexample, the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle). At leastone active agent in the composition is a Formula I compound. The labelor package insert indicates that the composition is used for treatingthe condition of choice, such as cancer. In one embodiment, the label orpackage inserts indicates that the composition comprising a Formula Icompound can be used to treat a disorder resulting from abnormal cellgrowth. The label or package insert may also indicate that thecomposition can be used to treat other disorders. Alternatively, oradditionally, the article of manufacture may further comprise a secondcontainer comprising a pharmaceutically acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

The kit may further comprise directions for the administration of thecompound of Formula I and, if present, the second pharmaceuticalformulation. For example, if the kit comprises a first compositioncomprising a compound of Formula I and a second pharmaceuticalformulation, the kit may further comprise directions for thesimultaneous, sequential or separate administration of the first andsecond pharmaceutical compositions to a patient in need thereof.

In another embodiment, the kits are suitable for the delivery of solidoral forms of a compound of Formula I, such as tablets or capsules. Sucha kit preferably includes a number of unit dosages. Such kits caninclude a card having the dosages oriented in the order of theirintended use. An example of such a kit is a “blister pack”. Blisterpacks are well known in the packaging industry and are widely used forpackaging pharmaceutical unit dosage forms. If desired, a memory aid canbe provided, for example in the form of numbers, letters, or othermarkings or with a calendar insert, designating the days in thetreatment schedule in which the dosages can be administered.

According to one embodiment, a kit may comprise (a) a first containerwith a compound of Formula I contained therein; and optionally (b) asecond container with a second pharmaceutical formulation containedtherein, wherein the second pharmaceutical formulation comprises asecond compound with anti-hyperproliferative activity. Alternatively, oradditionally, the kit may further comprise a third container comprisinga pharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

Where the kit comprises a composition of Formula I and a secondtherapeutic agent, i.e. the chemotherapeutic agent, the kit may comprisea container for containing the separate compositions such as a dividedbottle or a divided foil packet, however, the separate compositions mayalso be contained within a single, undivided container. Typically, thekit comprises directions for the administration of the separatecomponents. The kit form is particularly advantageous when the separatecomponents are preferably administered in different dosage forms (e.g.,oral and parenteral), are administered at different dosage intervals, orwhen titration of the individual components of the combination isdesired by the prescribing physician.

General Preparative Procedures

General Procedure A-1

Suzuki Coupling:

The Suzuki-type coupling reaction is useful to attach a fused bicyclicheterocycle or heteroaryl at the 2-position of the pyrimidine ring (seeScheme 4). Generally, substituted2-chloro-4-morpholinothieno[3,2-d]pyrimidine 5 may be combined with 1.5equivalents of4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)1H-indazole 7, anddissolved in 3 equivalents of sodium carbonate as a 1 molar solution inwater and an equal volume of acetonitrile. Intermediate 7 was preparedaccording to the methods of US 2008/0039459; US 2008/0076768; US2008/0076758; US 2008/0207611, incorporated by reference herein. Acatalytic amount, or more, of a low valent palladium reagent, such asbis(triphenylphosphine)palladium(II) dichloride, is added. A variety ofboronic acids or boronic esters can be used in place of the indazoleboronic ester indicated. Also alternatively, the nitrogen of theindazole may be protected, for example with a tetrahydropyranyl group.In some cases potassium acetate was used in place of sodium carbonate toadjust the pH of the aqueous layer. The reaction was then heated toabout 140-150° C. under pressure in a Biotage Optimizer microwavereactor (Biotage, Inc.) for 10 to 30 minutes. The contents are extractedwith ethyl acetate, or another organic solvent. After evaporation of theorganic layer the product 8 may be purified on silica or by reversephase HPLC.

General Procedure A-2

Suzuki Coupling:

The Suzuki-type coupling reaction is useful to attach a monocyclicheteroaryl at the 2-position of the pyrimidine ring (see Scheme 4).Generally, substituted 2-chloro-4-morpholinothieno[3,2-d]pyrimidine 5may be combined with 1.5 equivalents of5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine 7a, anddissolved in 3 equivalents of sodium or potassium carbonate as a 1 molarsolution in water and an equal volume of acetonitrile. Intermediate 7awas prepared according to the methods of US 2008/0269210; US2008/0242665, incorporated by reference herein. A catalytic amount, ormore, of a low valent palladium reagent, such asbis(triphenylphosphine)palladium(II) dichloride, is added. A variety ofboronic acids or boronic esters can be used in place of the pinacolboronic ester indicated. Also alternatively, the nitrogen of thepyrimidin-2-amine may be protected, for example with a tetrahydropyranylgroup. In some cases potassium acetate was used in place of sodiumcarbonate to adjust the pH of the aqueous layer. The reaction was thenheated, for example to about 100-150° C. under pressure in a BiotageOptimizer microwave reactor (Biotage, Inc.) for 10 to 30 minutes. Thecontents are extracted with ethyl acetate, or another organic solvent.After evaporation of the organic layer the product 8a may be purified onsilica or by reverse phase HPLC.

General Procedure B

Amide Coupling:

2-(1H-Indazol-4-yl)-4-morpholinothieno[3,2-d]pyrimidine-6-carboxylicacid 13 is treated with 1.5 eq HATU, 3 eq of alkylamine and 3 eq ofDIPEA in DMF to approximately 0.1 M concentration. The reaction isstirred until complete and extracted in ethylacetate with saturatedbicarbonate solution one time. The organic layer is dried, filtered andconcentrated to yield the crude intermediate. This intermediate ispurified via reverse phase HPLC to yield product 15.

General Procedure B-1

Amide Coupling:

4-Morpholino-2-(pyridin-3-yl)thieno[3,2-d]pyrimidine-6-carboxylic acid13a is treated with 1.5 eq HATU, 3 eq of an alkylamine (R—NH₂) and 3 eqof DIPEA in DMF to approximately 0.1 M concentration. The reaction isstirred until complete and extracted in ethylacetate with saturatedbicarbonate solution one time. The organic layer is dried, filtered andconcentrated to yield the crude intermediate. This intermediate ispurified via reverse phase HPLC to yield product 15a.

General Procedure B-2

Amide Coupling:

2-Chloro-4-morpholino-6-((piperazin-1-yl)methyl)thieno[3,2-d]pyrimidineis treated with about 1.5 eq of a coupling reagent such as HATU, about 3eq of a carboxylic acid (RCO₂H) and an excess of an amine base such asDIPEA in DMF to approximately 0.1 M concentration. The reaction isstirred until complete and extracted in ethyl acetate with saturatedbicarbonate solution one time. The organic layer is dried, filtered andconcentrated to yield the crude intermediate.

General Procedure B-3

Reductive Amination:

2-Chloro-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde is dissolvedto a 0.2 M concentration in dichloroethane. To this solution is added1.5 to 2.0 equivalents of a primary or secondary amine (R₂NH), about 10equivalents of trimethylorthoformate, and about 1 equivalent of aceticacid. The mixture is allowed to stir for 2-6 hours prior to adding 1.5equivalents of sodium triacetoxyborohydride. Following 12 to 16 hours ofstirring the reaction was poured into saturated sodium bicarbonate andextracted several times with ethyl acetate to give the reductiveamination intermediate which is either purified on silica gel or usedcrude in the next reaction.

General Procedure C

Sulfonamide Formation:

2-Chloro-4-morpholinothieno[3,2-d]pyrimidine-6-sulfonyl chloride 17 issuspended in DCM before addition of about 2 eq of amine (HNR₂) and about3 eq of an amine base such as DIPEA. The reactions are monitored by LCMSuntil complete. The crude reaction mixtures are diluted with ethylacetate, extracted with saturated ammonium chloride and back-extractedonce with ethyl acetate. The organic layers were combined andconcentrated to dryness. The crude sulfonamide intermediates 18 are useddirectly in the subsequent Suzuki couplings.

General Procedure D

Alcohol Synthesis

2-Chloro-4-morpholinothieno[3,2-d]pyrimidine 4 is suspended to a 0.2molar concentration in THF and cooled to −50° C. in a dryice/acetonitrile bath before adding 2 equivalents of 2.5 M nBuLi inhexanes. After 15 min 3.0 molar equivalents of a cyclic or acyclicketone (R₂C(═O) is added to the solution. The reaction continued to stirat −50° C. for 1 h and then in most cases was allowed to come to 0° C.When the reaction is complete by TLC or mass spec. it is quenched into asaturated ammonium chloride solution and extracted two times with EtOAc.The organic layer is concentrated and either used as a crude mixture,purified on silica, or the product 12 could be dissolved in a minimalamount of acetonitrile and filtered to remove remaining startingmaterial 4.

General Procedure E

Removal of t-butoxylcarbonyl (BOC) Group

Ten or more equivalents of 4N HCl in dioxane, with or withoutdichloromethane as a co-solvent, are added to the starting material(general scheme shown above but similar scaffolds also used). Heating upto 40° C. for several hours is occasionally required to remove the Bocgroup. The reaction is concentrated to dryness and may be used crude insubsequent reactions.

General Procedure F

Suzuki Coupling Reactions in One Pot

2-Chloro-6-iodo-4-morpholinothieno[3,2-d]pyrimidine 19 (1 eq),phenylboronic acid or heterocycleboronic acid (R¹—B(OH)₂, 1.1 eq) andbis(triphenylphosphine)palladium(II) dichloride (0.1 eq) in 1M Na₂CO₃aqueous solution (3 eq) and acetonitrile (3 eq) was heated to 100° C. ina sealed microwave reactor for 10 to 40 min to give 5. Upon completion,4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole 7 (1.3 eq)and bis(triphenylphosphine)palladium(II) dichloride (0.1 eq) were addedin the same pot. The reaction mixture was heated to 150° C. in a sealedmicrowave reactor for 10 to 15 min. The mixture was extracted with ethylacetate (3×5 mL). The combined organic layers were concentrated to yieldcrude 8.

General Procedure G

Amide Coupling Reaction

2-Chloro-4-morpholinothieno[3,2-d]pyrimidin-6-amine 22 (1 eq), acidchloride (about 2 eq) and triethylamine (2 eq) in dichloromethane wasstirred. The reaction was monitored by LC/MS until complete. The mixturewas evaporated to give the crude amide 23, which was directly used forthe next step reaction without purification.

General Procedure H

Preparation of Acetamide, Benzamidines, and Sulfonamides

To a 0.25 to 0.40 M solution of1-(2-chloro-4-morpholinothieno[2,3-d]pyrimidin-6-yl)-N-methylmethanaminein DCM cooled to 0° C. was added 1.5 eq. of TEA, followed by thedrop-wise addition of 1.0 to 1.5 eq. of an alkyl or aryl-acid chlorideor a sulfonylchloride, diluted in DCM. The reaction is stirred atambient temperature and monitored for completeness by LCMS. Aftercompletion, the reaction volume is increased with DCM, and diluteaqueous sodium bicarbonate is added to the solution. The organic andaqueous layers are separated. Finally, the organic layer is washed withbrine and dried (MgSO₄). The dried organic solution is concentrated invacuo and the product is purified by silica chromatography if necessary.

General Procedure I

Amide Coupling Reaction for Benzenamine

3-(2-Chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)benzenamine 24 (1eq), carboxylic acid (1.5 eq), 1-hydroxy-7-azabenzotriazole (0.2 eq),O-(7-azabenzotriazol-1-yl)-(N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU, 1.5 eq), and N,N-diisopropylethylamine (2.5eq) in DMF was stirred at room temperature. The reaction was monitoredby LC/MS until complete. The reaction mixture was diluted with ethylacetate, washed with saturated sodium bicarbonate and brine. The organiclayer was dried over MgSO₄, filtered and evaporated to yield amideproduct 25.

General Procedure J

6-Iodo Displacement and 2-Suzuki Coupling

To a solution of 2-chloro-6-iodo-4-morpholinothieno[3,2-d]pyrimidine 19(0.05 g, 0.13 mmol) in DMF (1.00 mL) was added the appropriate aniline(200 mol %), Cs—₂CO₃ (50 mol %), Pd₂(dba)₃ (5 mol %), and XANTPHOS (10mol %). The reaction was heated to 110° C. under pressure in a Biotageoptimizer microwave reactor for 30 min. The resulting solution wasconcentrated in vacuo to give 26, after following General Procedure A.

General Procedure K

6-Aminoalkyl Acylation and 2-Suzuki Coupling

To a solution of(2-chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methanamine 27 (50 mg,0.2 mmol) in CH₂Cl₂ (4 mL) was added Et₃N (84 μL, 0.6 mmol) and theappropriate acid chloride or HCl salt thereof (0.3 mmol). The reactionstirred 18-48 hr at room temperature before being quenched with water.The aqueous layer was extracted with EtOAc. The combined organics weredried over Na₂SO₄ and concentrated in vacuo. The 2-chloro crude productwas coupled with boronate reagent 7 and palladium catalyst according toGeneral Procedure A to give 28 which was purified by reversed phase HPLCpurification.

Alternatively, to a solution of(2-chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methanamine 27 (111mg, 0.39 mmol) in DMF (5 mL) was added 2,6-lutidine (48.2 μL, 0.41 mmol)and the appropriate acid chloride or HCl salt thereof (0.39 mmol). Thereaction stirred 18-72 hr at room temperature before being quenched withwater. The aqueous layer was extracted with EtOAc. The combined organicswere dried over MgSO₄ and concentrated in vacuo. The 2-chloro crudeproduct was coupled with boronate reagent 7 and palladium catalystaccording to General Procedure A to give 20 mg of 28 which was purifiedby reversed phase HPLC purification.

General Procedure L

Amine Substitution on Fluoropyridine

A mixture of2-chloro-6-(6-fluoropyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidine,about four equivalents of a primary or secondary amine (R═H, C₁-C₁₂alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₁₂ carbocyclyl, C₂-C₂₀heterocyclyl, C₆-C₂₀ aryl, or C₁-C₂₀ heteroaryl), and about two eq.diisopropylethylamine in N-methylpyrrolidine (˜0.1M) is heated to about130-140° C. in a sealed microwave reactor for 10-40 min, followed byremoval of volatiles under high vacuum. The crude mixture is purified byflash chromatography to give intermediate2-chloro-6-(6-aminopyridin-3-yl)-4-morpholinothieno[3,2-d]pyrimidine,which may be Suzuki coupled with a monocyclic heteroaryl, fused bicyclicheterocycle or heteroaryl boronate reagent following General ProcedureA.

EXAMPLES

In order to illustrate the invention, the following examples areincluded. However, it is to be understood that these examples do notlimit the invention and are only meant to suggest a method of practicingthe invention. Persons skilled in the art will recognize that thechemical reactions described may be readily adapted to prepare a numberof other PI3K inhibitors of the invention, and alternative methods forpreparing the compounds of this invention are deemed to be within thescope of this invention. For example, the synthesis of non-exemplifiedcompounds according to the invention may be successfully performed bymodifications apparent to those skilled in the art, e.g., byappropriately protecting interfering groups, by utilizing other suitablereagents known in the art other than those described, and/or by makingroutine modifications of reaction conditions. Alternatively, otherreactions disclosed herein or known in the art will be recognized ashaving applicability for preparing other compounds of the invention.

Example 1 2,4-Dichloro-thieno[3,2-d]pyrimidine 3

A mixture of methyl 3-amino-2-thiophenecarboxylate 1 (13.48 g, 85.85mmol) and urea (29.75 g, 5 eq.) was heated at 190° C. for 2 hours. Thehot reaction mixture was poured onto sodium hydroxide solution and anyinsoluble material was removed by filtration. The mixture was thenacidified (HCl, 2N) to yield 1H-thieno[3,2-d]pyrimidine-2,4-dione 2 as awhite precipitate, which was collected by filtration and air dried (9.49g, 66%). ¹H NMR 400 MHz, d₆-DMSO) 6.90 (1H, d, J=5.2 Hz), 8.10 (1H, d,J=5.2 Hz), 11.60-11.10 (2H, br s).

A mixture of 1H-thieno[3,2-d]pyrimidine-2,4-dione 2 (9.49 g, 56.49 mmol)and phosphorous oxychloride (150 mL) was heated at reflux for 6 h. Thereaction mixture was then cooled and poured onto ice/water with vigorousstirring yielding a precipitate. The mixture was then filtered to yield2,4-dichloro-thieno[3,2-d]pyrimidine 3 as a white solid (8.68 g, 75%). HNMR (400 MHz, CDCl₃) 7.56 (1H, d, J=5.5 Hz), 8.13 (1H, d, J=5.5 Hz).

Example 2 2-Chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine 4

A mixture of 2,4-dichloro-thieno[3,2-d]pyrimidine 3, (8.68 g, 42.34mmol), morpholine (8.11 mL, 2.2 eq.) and MeOH (150 mL) was stirred atroom temperature for 1 h. The reaction mixture was then filtered, washedwith water and MeOH, to yield2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine 4 as a white solid(11.04 g, 100%). ¹H NMR (400 MHz, d₆-DMSO) 3.74 (4H, t, J=4.9 Hz), 3.90(4H, t, J=4.9 Hz), 7.40 (1H, d, J=5.6 Hz), 8.30 (1H, d, J=5.6 Hz).

Example 32-Chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde 10

To a suspension of 2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine 4(1.75 g, 6.85 mmol) in dry THF (40 mL) at −78° C. was added a 2.5Msolution of n-butyllithium (nBuLi) in hexane (3.3 mL, 1.2 eq.). Afterstirring for 1 h, dry DMF (796 μL, 1.5 eq.) was added. The reactionmixture was stirred for 1 h at −78° C. and then warmed slowly to roomtemperature. After a further 2 h at room temperature the reactionmixture poured onto ice/water yielding a yellow precipitate. This wascollected by filtration and air-dried to yield2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde 10(1.50 g, 77%). ¹H NMR (400 MHz, d₆-DMSO) 3.76 (4H, t, J=4.9), 3.95 (4H,t, J=4.9), 8.28 (1H, s), 10.20 (1H, s).

Example 4 2-Chloro-6-iodo-7-methyl-4-morpholinothieno[3,2-d]pyrimidine41

To a solution of 2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidine39 (3.0 g, 11.1 mmol; prepared according to the procedure for thesynthesis of 2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine butcommencing with 3-amino-4-methyl-thiophene-2-carboxylic acid ethylester) in THF (60 mL) at −78° C. was added n-BuLi (8.9 mL, 2.5 M inEt₂O). The resulting slurry was warmed to −40° C. and stirred 50 min.The reaction mixture was then cooled to −78° C. and a solution of I₂(5.6 g, 22.2 mmol) in THF (30 mL) was added. The solution was warmed toroom temperature and stirred 5 h. The reaction was quenched by theaddition of water. The organic layer was separated and the aqueous layerwas extracted with CH₂Cl₂. The combined organics were washed withsaturated aqueous Na₂S₂O₃, dried over Na₂SO₄, filtered, and concentratedin vacuo to provide2-chloro-6-iodo-7-methyl-4-morpholinothieno[3,2-d]pyrimidine 41 (3.8 g,84% yield).

Example 54-(2-Chloro-6-(piperazin-1-ylmethyl)thieno[3,2-d]pyrimidin-4-yl)morpholine30

A mixture of2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde 10 (3.5g), 1-BOC-piperazine (2.76 g) and trimethylorthoformate (4.05 mL) wasstirred in 1,2-dichloroethane (300 mL) for 1 hr at room temperature. Tothis was added sodium triacetoxyborohydride (3.92 g) and the reactionmixture was stirred for 24 hours at room temperature. The mixture wasthen quenched with brine, extracted with dichloromethane, dried (MgSO₄)and the solvent removed in vacuo. The residue was purified using flashchromatography to yield4-(2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidin-6-ylmethyl)-piperazine-1-carboxylicacid tert-butyl ester (3.4 g). Treatment with HCl indichloromethane/methanol yielded4-(2-chloro-6-(piperazin-1-ylmethyl)thieno[3,2-d]pyrimidin-4-yl)morpholine30.

Example 6(2-chloro-4-morpholinothieno[3,2-d]pyrimidin-6-yl)-N-methylmethanamine35

2-Chloro-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde 10 (2.0 g)was dissolved in 50 mL toluene and 50 mL THF followed by the addition of20 mL of 40% methylamine in H₂O. The reaction mixture was stirred atroom temp under N₂ for 24 hours. The solvents were removed in vacuo andthe residue was dissolved in 50 mL methanol and 50 mL THF and the NaBH₄added portion-wise. This reaction mixture was stirred at room temp underN₂ for 24 hours and complete reaction was confirmed by LCMS. Thesolvents were removed in vacuo and the crude product purified by flashchromatography (EtOAc/EtOH) to give 1.12 g 35 (53% yield). MS (Q1) 300(M+).

Example 7(2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)-N-methylmethanamine37

2-Chloro-7-methyl-4-morpholinothieno-[3,2-d]pyrimidine-6-carbaldehyde 36was dissolved in 20 mL toluene and 20 mL THF followed by the addition of15 mL 40% methylamine in H₂O and the reaction was stirred for 24 hours.The reaction mixture was concentrated in vacuo and the residue dissolvedin 30 mL methanol and 30 mL THF followed by the addition of NaBH₄. Thereaction was stirred at room temp for at least 24 hours and productformation was confirmed by LCMS. The solvents were removed in vacuo andthe crude product purified by flash chromatography to give 2.53 g(2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)-N-methylmethanamine37 (70% yield) MS (Q1) 314 (M)+

Example 84-(2-Chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine31

Reaction between N—BOC-piperazine and methane sulfonyl chloride indichloromethane and triethylamine yielded4-methanesulfonyl-piperazine-1-carboxylic acid tert-butyl ester.Cleavage of the BOC protecting group using HCl (2M) in dichloromethaneyielded 1-methanesulfonyl-piperazine HCl salt.

A mixture of2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde 10(1.00 g), 1-methanesulfonyl-piperazine (750 mg) andtrimethylorthoformate (3.80 mL) was stirred in 1,2-dichloroethane (30mL) for 6 hrs at room temperature. To this was added sodiumtriacetoxyborohydride (900 mg) and the reaction mixture was stirred for24 hours at room temperature. The mixture was then quenched with brine,extracted with dichloromethane, dried (MgSO₄) and the solvent removed invacuo. The residue was triturated with hot ethyl acetate to yield4-(2-chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine31 as a white solid (1.01 g).

Example 9 4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole7-route 1

Intermediate 7 was prepared according to the methods of US 2008/0076768;US 2008/0076758; WO 2006/046031, incorporated by reference herein.

Example 101-(Tetrahydro-2H-pyran-2-yl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole40

Intermediate 40 was prepared according to the methods of US2008/0039459; US 2008/0076768; US 2008/0076758; US 2008/0207611,incorporated by reference herein.

Example 112-(1H-Indazol-4-yl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde11

A mixture of2-chloro-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde 10 (100mg, 0.35 mmol),4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole (70) (95mg, 0.39 mmol) and sodium carbonate (112 mg) were suspended in toluene(2.5 mL), ethanol (1.5 mL) and water (0.7 mL). To this was addedbis(triphenylphosphine)palladium(II) chloride (13.5 mg) and the reactionvessel was flushed with argon. The reaction mixture was microwaved at120° C. for 1 h and then partitioned between DCM and water, the organiclayer was washed with brine, dried over magnesium sulfate, filtered andevaporated in vacuo. The resulting residue was purified using flashchromatography to yield2-(1H-indazol-4-yl)-4-morpholin-4-yl-thieno[3,2-d]pyrimidine-6-carbaldehyde11 (97 mg).

Example 124-(2-(1H-Indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine(Formula Ia, GDC-0941)

A mixture of4-(2-chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4-yl)morpholine31 from Example 4 (2.00 g),4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-1H-indazole 7 (2.26 g),toluene (24 mL), ethanol (12 mL), water (6 mL), sodium carbonate (1.72g) and PdCl₂(PPh₃)₂ (325 mg) was heated to 130° C. in the microwave for90 minutes (US 2008/0076768; WO 2006/046031, incorporated by referenceherein).

The reaction mixture was cooled, diluted with chloroform, washed withbrine, dried (MgSO₄) and the solvent removed in vacuo. The residue waspurified using flash chromatography (ethyl acetate then 5% ethylacetate/methanol) and then trituration with ether yielded Formula Iacompound, GDC-0941 (1.4 g). MS data: (ESI+): MH+ 514. NMR data: (CDCl₃):2.67-2.71 (4H, m), 2.81 (3H, s), 3.29-3.33 (4H, m), 3.89 (2H, s),3.89-3.93 (4H, m), 4.08-4.12 (4H, m), 7.41 (1H, s), 7.51 (1H, t, J=7.2),7.60 (1H, d, J=8.3), 8.28 (1H, d, J=7.5), 9.02 (1H, s), 10.10 (1H, br)

Example 13(S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one(Formula Ib)

2-Chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidine-6-carbaldehyde 36(495 mg) was reacted with Boc-piperazine via General Procedure B-3 togive tert-butyl4-((2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazine-1-carboxylate.

Tert-butyl4-((2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazine-1-carboxylate(777 mg) was subjected to General Procedure E to give the HCl salt of2-chloro-7-methyl-4-morpholino-6-((piperazin-1-yl)methyl)thieno[3,2-d]pyrimidine.The HCl salt of2-chloro-7-methyl-4-morpholino-6-((piperazin-1-yl)methyl)thieno[3,2-d]pyrimidine(590 mg) was reacted with lactic acid via General Procedure B-2 to give(S)-1-(4-((2-chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one.

(S)-1-(4-((2-Chloro-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one(60 mg) was reacted with 50 mg of5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine viaGeneral Procedure A-2 to give 10 mg of Formula Ib (US 2008/0242665,incorporated by reference). MS (Q1) 499.3 (M)+.

Example 14 p110α (alpha) PI3K Binding Assay

Binding Assays: Initial polarization experiments were performed on anAnalyst HT 96-384 (Molecular Devices Corp, Sunnyvale, Calif.). Samplesfor fluorescence polarization affinity measurements were prepared byaddition of 1:3 serial dilutions of p110alpha PI3K (Upstate CellSignaling Solutions, Charlottesville, Va.) starting at a finalconcentration of 20 ug/mL in polarization buffer (10 mM Tris pH 7.5, 50mM NaCl, 4 mM MgCl₂, 0.05% Chaps, and 1 mM DTT) to 10 mM PIP₂(Echelon-Inc., Salt Lake City, Utah) final concentration. After anincubation time of 30 minutes at room temperature, the reactions werestopped by the addition of GRP-1 and PIP3-TAMRA probe (Echelon-Inc.,Salt Lake City, Utah) 100 nM and 5 nM final concentrations respectively.Read with standard cut-off filters for the rhodamine fluorophore(λex=530 nm; λem=590 nm) in 384-well black low volume Proxiplates(PerkinElmer, Wellesley, Mass.) Fluorescence polarization values wereplotted as a function of the protein concentration, and the EC₅₀ valueswere obtained by fitting the data to a 4-parameter equation usingKaleidaGraph software (Synergy software, Reading, Pa.). This experimentalso establishes the appropriate protein concentration to use insubsequent competition experiments with inhibitors.

Inhibitor IC₅₀ values were determined by addition of the 0.04 mg/mLp110alpha PI3K (final concentration) combined with PIP₂ (10 mM finalconcentration) to wells containing 1:3 serial dilutions of theantagonists in a final concentration of 25 mM ATP (Cell SignalingTechnology, Inc., Danvers, Mass.) in the polarization buffer. After anincubation time of 30 minutes at room temperature, the reactions werestopped by the addition of GRP-1 and PIP3-TAMRA probe (Echelon-Inc.,Salt Lake City, Utah) 100 nM and 5 nM final concentrations respectively.Read with standard cut-off filters for the rhodamine fluorophore(λex=530 nm; λem=590 nm) in 384-well black low volume proxi plates(PerkinElmer, Wellesley, Mass.) Fluorescence polarization values wereplotted as a function of the antagonist concentration, and the IC₅₀values were obtained by fitting the data to a 4-parameter equation inAssay Explorer software (MDL, San Ramon, Calif.).

Alternatively, inhibition of PI3K was determined in a radiometric assayusing purified, recombinant enzyme and ATP at a concentration of 1 uM.The compound was serially diluted in 100% DMSO. The kinase reaction wasincubated for 1 h at room temperature, and the reaction was terminatedby the addition of PBS. IC₅₀ values were subsequently determined usingsigmoidal dose-response curve fit (variable slope).

Example 15 In Vitro Cell Proliferation Assay

Efficacy of Formula I or II compounds were measured by a cellproliferation assay employing the following protocol (Mendoza et al(2002) Cancer Res. 62:5485-5488). The CellTiter-Glo® Luminescent CellViability Assay, including reagents and protocol are commerciallyavailable (Promega Corp., Madison, Wis., Technical Bulletin TB288). Theassay assesses the ability of compounds to enter cells and inhibit cellproliferation. The assay principle is based on the determination of thenumber of viable cells present by quantitating the ATP present in ahomogenous assay where addition of the Cell-Titer Glo reagent results incell lysis and generation of a luminescent signal through the luciferasereaction. The luminescent signal is proportional to the amount of ATPpresent.

Procedure: Day 1—Seed Cell Plates (384-well black, clear bottom,microclear, TC plates with lid from Falcon #353962), Harvest cells, Seedcells at 1000 cells per 54 μl per well into 384 well Cell Plates for 3days assay. Cell Culture Medium: RPMI or DMEM high glucose, 10% FetalBovine Serum, 2 mM L-Glutamine, P/S. Incubate O/N at 37 C, 5% CO2.

Day 2—Add Drug to Cells, Compound Dilution, DMSO Plates (serial 1:2 for9 points), Add 20 ul compounds at 10 mM in the 2nd column of 96 wellplate. Perform serial 1:2 across the plate (10 μl+20 μl 100% DMSO) for atotal of 9 points using Precision. Media Plates 96-well conical bottompolypropylene plates from Nunc (cat. #249946) (1:50 dilution) Add 147 μlof Media into all wells. Transfer 3 μl of DMSO+compound from each wellin the DMSO Plate to each corresponding well on Media Plate usingRapidplate. For 2 drug combo studies, transfer one drug 1.5 μl ofDMSO+compound from each well in the DMSO Plate to each correspondingwell on Media Plate using Rapidplate. Then, transfer another drug 1.5 ulto the medium plate.

Drug Addition to Cells, Cell Plate (1:10 dilution), Add 6 μl ofmedia+compound directly to cells (54 μl of media on the cells already).Incubate 3 days at 37 C, 5% CO2 in an incubator that will not be openedoften.

Day 5—Develop Plates, Thaw Cell Titer Glo Buffer at room temperature.Remove Cell Plates from 37° C. and equilibrate to room temperature. forabout 30 minutes. Add Cell Titer Glo Buffer to Cell Titer Glo Substrate(bottle to bottle). Add 30 μl Cell Titer Glo Reagent (Promega cat.#G7572) to each well of cells. Place on plate shaker for about 30minutes. Read luminescence on Analyst HT Plate Reader (half second perwell).

Cell viability assays and combination assays: Cells were seeded at1000-2000 cells/well in 384-well plates for 16 h. On day two, nineserial 1:2 compound dilutions were made in DMSO in a 96 well plate. Thecompounds were further diluted into growth media using a Rapidplaterobot (Zymark Corp., Hopkinton, Mass.). The diluted compounds were thenadded to quadruplicate wells in 384-well cell plates and incubated at 37C and 5% CO2. After 4 days, relative numbers of viable cells weremeasured by luminescence using Cell-Titer Glo (Promega) according to themanufacturer's instructions and read on a Wallac Multilabel Reader(PerkinElmer, Foster City). EC50 values were calculated using Prism 4.0software (GraphPad, San Diego). Drugs in combination assays were dosedstarting at 4×EC50 concentrations. If cases where the EC50 of the drugwas >2.5 μM, the highest concentration used was 10 μM. PI3K inhibitorsand chemotherapeutic agents were added simultaneously or separated by 4hours (one before the other) in all assays.

An additional exemplary in vitro cell proliferation assay includes thefollowing steps:

1. An aliquot of 100 μl of cell culture containing about 10⁴ cells (seeFIGS. 1A-C for cell lines and tumor type) in medium was deposited ineach well of a 384-well, opaque-walled plate.

2. Control wells were prepared containing medium and without cells.

3. The compound was added to the experimental wells and incubated for3-5 days.

4. The plates were equilibrated to room temperature for approximately 30minutes.

5. A volume of CellTiter-Glo Reagent equal to the volume of cell culturemedium present in each well was added.

6. The contents were mixed for 2 minutes on an orbital shaker to inducecell lysis.

7. The plate was incubated at room temperature for 10 minutes tostabilize the luminescence signal.

8. Luminescence was recorded and reported in graphs as RLU=relativeluminescence units.

9. Analyze using the Chou and Talalay combination method and Dose-EffectAnalysis with CalcuSyn software (Biosoft, Cambridge, UK) in order toobtain a Combination Index.

Alternatively, cells were seeded at optimal density in a 96 well plateand incubated for 4 days in the presence of test compound. Alamar Blue™was subsequently added to the assay medium, and cells were incubated for6 h before reading at 544 nm excitation, 590 nm emission. EC₅₀ valueswere calculated using a sigmoidal dose response curve fit.

Alternatively, Proliferation/Viability was analyzed after 48 hr of drugtreatment using Cell Titer glo reagent (Promega Inc., Madison, Wis.).DMSO treatment was used as control in all viability assays. IC₅₀ valueswere calculated using XL fit software (IDBS, Alameda, Calif.)

The AML cell lines AP-1060, EOL-1, FKH-1, GF-D8, HEL, HL-60, HNT-34,Kasumi-1, KG-1, ME-1, ML-2, MOLM-13, MOLM-16, MV4-11, NOMO-1, OCI-AML2,OCI-AML3, OCI-AML5, OCI-M1, OCI-M2, PL-21, SET-2, SIG-M5, SKM-1, THP-1,UKE-1, and UT-7 were obtained from either ATCC or DSMZ. Cells aremaintained in RPMI 1640 media with 10% heat-inactivated FBS(Sigman-Aldrich) and 2 mol/L L glutamine. Antibodies for Westernblotting were purchased from Cell Signal Technology, with the exceptionof p110δ antibody which was obtained from Epitomics. Inhibitor GDC-0941was synthesized at Genentech. Rapamycin, Ara-C, and daunorubicin werepurchased from Sigma. All compounds were prepared as stock solutions inDMSO, and diluted in assay buffer upon use. Blast cells were isolatedfrom AML patient peripheral blood or bone marrow samples by Ficollseparation. After wash in PBS, isolated cells were cultured in RPMI 1640media with 10% FBS and a cytokine cocktail as indicated (IGF-1, SCF,GMCSF, and IL-3 at 10 ng/ml each).

Human multiple myeloma cell lines were all obtained from the AmericanType Culture Collection (ATCC, Manassas, Va.) and also mentioned inearlier studies( ). Cells were cultured in RPMI 1640 medium supplementedwith 10% fetal bovine serum, 100 units/ml penicillin, 2 mM L-glutamine,and 100 mg/ml streptomycin (Life Technology, Grand Island, N.Y.) at 37°C. under 5% CO₂.

Example 16 In Vivo Mouse Tumor Xenograft Efficacy

Mice: Female severe combined immunodeficiency mice (Fox Chase SCID®,C.B-17/IcrHsd, Harlan) were 8 to 9 weeks old and had a BW range of 15.1to 21.4 grams on Day 0 of the study. The animals were fed ad libitumwater (reverse osmosis, 1 ppm Cl) and NIH 31 Modified and Irradiated LabDiet® consisting of 18.0% crude protein, 5.0% crude fat, and 5.0% crudefiber. The mice were housed on irradiated ALPHA-Dri® Bed-O'Cobs®Laboratory Animal Bedding in static microisolators on a 12-hour lightcycle at 21-22° C. (70-72° F.) and 40-60% humidity. PRC specificallycomplies with the recommendations of the Guide for Care and Use ofLaboratory Animals with respect to restraint, husbandry, surgicalprocedures, feed and fluid regulation, and veterinary care. The animalcare and use program at PRC is accredited by the Association forAssessment and Accreditation of Laboratory Animal Care International(AAALAC), which assures compliance with accepted standards for the careand use of laboratory animals.

Tumor Implantation:

Xenografts were initiated with cancer cells. Cells were cultured in RPMI1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine,100 units/mL penicillin, 100 g/mL streptomycin sulfate and 25 g/mLgentamicin. The cells were harvested during exponential growth andresuspended in phosphate buffered saline (PBS) at a concentration of5×10⁷ cells/mL. On the day of tumor implant, each test mouse received1×10^7 cells (0.2 mL) implanted subcutaneously in the right flank, andtumor growth was monitored as the average size approached the targetrange of 100 to 150 mm3. Twenty-one days after tumor implantation,designated as Day 0 of the study, the mice were placed into four groupseach consisting of ten mice with individual tumor volumes ranging from75-172 mm3 and group mean tumor volumes from 120-121 mm3 (see AppendixA). Volume was calculated using the formula:Tumor Volume (mm³)=(w ² ×l)/2

where w=width and l=length in mm of a tumor. Tumor weight may beestimated with the assumption that 1 mg is equivalent to 1 mm3 of tumorvolume.

Therapeutic Agents:

Formula Ia compound (GDC-0941, Genentech, Inc.) was supplied as a drypowder in salt form, which contained 73% active agent, and was stored atroom temperature protected from light. Drug doses were prepared weeklyin 0.5% methylcellulose: 0.2% Tween 80 in deionized water (MC/Tw80,“Vehicle”) and stored at 4° C. The salt form containing 73% active agentwas accounted for in the formulation of G-033829 doses. Rituximab(Rituxan® 10 mg/mL for injection, Genentech, Lot #M79901) was purchasedas the clinical drug. Doses of rituximab were prepared on each day ofdosing by diluting an aliquot of the stock with sterile saline (0.9%NaCl). All doses were formulated to deliver the stated mg/kg dosage in avolume of 0.2 mL per 20 grams of body weight (10 mL/kg).

Treatment:

All doses were scaled to the body weights of the individual animals andwere provided by the route indicated in each of the figures.

Endpoint:

Tumors were measured twice each week using calipers. Mice were monitoredindividually, and each animal was euthanized when its tumor reached avolume of 1500 mm3 or at the end of the study on Day 40, whichever camefirst. However, due to the self-limiting growth of control tumors, theendpoint was reduced to 1000 mm3 for analysis. The time to endpoint(TTE) for each mouse was calculated from the following equation:TTE (days)=[log₁₀(endpoint volume,mm³)−b]/m

where b is the intercept and m is the slope of the line obtained bylinear regression of a log-transformed tumor growth data set. The dataset was comprised of the first observation that exceeded the studyendpoint volume and the three consecutive observations that immediatelypreceded the attainment of the endpoint volume. Animals that do notreach the endpoint are assigned a TTE value equal to the last day of thestudy. Animals classified as NTR (non-treatment-related) deaths due toaccident (NTRa) or due unknown causes (NTRu) are excluded from TTEcalculations (and all further analyses). Animals classified as TR(treatment-related) deaths or NTRm (non-treatment-related death due tometastasis) are assigned a TTE value equal to the day of death.Treatment outcome was evaluated by tumor growth delay (TGD), which isdefined as the increase in the median time to endpoint (TTE) in atreatment group compared to the control group:TGD=T−C

expressed in days, or as a percentage of the median TTE of the controlgroup:% TGD=(T−C)/C×100

where: T=median TTE for a treatment group, C=median TTE for the controlgroup (Group 1). Treatment may cause partial regression (PR) or completeregression (CR) of the tumor in an animal. In a PR response, the tumorvolume is 50% or less of its Day 1 volume for three consecutivemeasurements during the course of the study, and equal to or greaterthan 13.5 mm3 for one or more of these three measurements. In a CRresponse, the tumor volume is less than 13.5 mm3 for three consecutivemeasurements during the course of the study. An animal with a CRresponse at the termination of a study is additionally classified as atumor-free survivor (TFS). Animals were monitored for regressionresponses.

Toxicity:

Animals were weighed daily for the first five days of the study andtwice weekly thereafter. The mice were observed frequently for overtsigns of any adverse, treatment-related side effects, and clinical signsof toxicity were recorded when observed. Acceptable toxicity is definedas a group mean body weight (BW) loss of less than 20% during the studyand not more than one treatment-related (TR) death among ten treatedanimals. Any dosing regimen that results in greater toxicity isconsidered above the maximum tolerated dose (MTD). A death is classifiedas TR if attributable to treatment side effects as evidenced by clinicalsigns and/or necropsy, or may also be classified as TR if due to unknowncauses during the dosing period or within 10 days of the last dose. Adeath is classified as NTR if there is no evidence that death wasrelated to treatment side effects.

Example 17 Western Blotting for Detection of p-Akt, p-BAD and p-S6Ribosomal Protein Post GDC-0941 Treatment of B Cell and Myeloma CellLines

Materials: All reagents for electrophoresis and blotting buffer stocksare from Invitrogen.

B cell lines (DoHH2 and WSU-DLCL2) and Myeloma cell lines (OPM2 andU266) were maintained in RPMI-1640/10% FBS in the presence of Pen/Strepand Glutamine.

Protocol:

-   1. Seed 10⁷ cells of each cell line in 10 mL culture media in 10 cm    Petri dish, 2 dishes for each cell line.-   2. Add 5 uL of 10 mM GDC-0941 stock (in DMSO) to one dish of 10 mL    culture for final 5 uM drug and add 5 uL DMSO to the other dish as    control.-   3. Keep cells at 37° C., 5% CO2 incubator for 4 hours before    harvesting.-   4. Harvest 9 mL culture (equivalent to 9×10⁶ cells) to 15 mL conical    tube and pellet and make cell lysates with 1×SDS sample buffer.    Transfer the other 1 mL of each condition (equivalent to 1×10⁶    cells) to 5 mL FACS tube (BD) for FACS.-   5. Wash cells once with cold PBS.-   6. Measure the OD of the protein samples and prepare the samples    with NuPAGE electrophoresis system reagents to load 20 ug of each.-   7. Mix each protein sample for electrophoresis with appropriate    amount of NuPAGE LDS sample buffer and Reducing Agent and apply    70° C. heat for 10 min.-   8. Load 10 uL of SeeBlue Plus2 Pre-stained standard and 20 ug of    each sample into 4-12% NuPAGE Norvex Bis-Tris Gel and run with    MES-SDS buffer in the presence of antioxidant (135 volts for 1 hour    in the XCell SureLock Mini-Cell apparatus).-   9. Transfer gels to the blotting membrane using iBlot apparatus.-   10. Wash 1× with 1×TBST buffer.-   11. Block the membrane with 20 mL 1×TBST 5% non-fat milk for 2    hours.-   12. Wash 3× with 1×TBST buffer.-   13. Incubate membrane in 10 mL 1×TBST 5% BSA and primary abs (p-Akt,    clone 193H12, p-BAD, clone 185D10 and p-S6RP, clone D57.2.2D,    beta-actin, clone 13E5, Cell Signaling) at appropriate dilution at 4    C O/N. p-Akt and p-6SRP and beta-actin rabbit abs are added to the    same membrane and others each on a separate membrane (multiple gels    are generated early).-   14. Wash 3× with 1×TBST buffer.-   15. Incubate membrane with HRP-conjugated Goat anti-rabbit ab at    appropriate dilution in 10 mL 1×TBST buffer/5% non-fat milk at room    temperature for 2 hours.-   16. Wash 3× with 1×TBST buffer.-   17. Incubate membrane with 3 mL LumiGLO for 3 min, drain the excess    solution and expose to x-ray film.

Alternatively, 10×10⁶ cells for each condition were plated in 10 cm²plate, and cells were treated using GDC-0941, dexamethasone, orlenalidomide, and or in combinations. DMSO treatment was used ascontrol. Cells were washed once with cold PBS and lysed using 1× celllysis buffer (Cell Signaling Technology, Beverly, Mass.). An equalamount of protein was resolved using Nupage Bis-Tris gels (Invitrogen,Carlsbad, Calif.). Western blot analysis was performed usinganti-phospho-Akt (Serine 473), anti-PTEN, anti-phospho-BAD (serine 136),anti-phospho-FoXO 1/3a, Bim, cleaved caspase 9, cleaved caspase 3, p27,and cleaved PARP antibodies (Cell Signaling Technology, Beverly, Mass.).Total AKT, total BAD, total Foxo3a and β-actin (Sigma) levels were usedas loading controls.

Example 18 FACS Protocol for Intracellular Detection of p-Akt and p-S6Ribosomal Protein Post GDC-0941 Treatment

B cell lines (DoHH2 and WSU-DLCL2) and Myeloma cell lines (OPM2 andU266) were maintained in RPMI-1640/10% FBS in the presence of Pen/Strepand Glutamine.

Protocol:

-   1. Seed 10⁷ cells of each cell line in 10 mL culture media in 10 cm    Petri dish, 2 dishes for each cell line.-   2. Add 5 uL of 10 mM GDC-0941 stock (in DMSO) to 10 mL culture for    final 5 uM drug and add 5 uL DMSO to the other dish as control.-   3. Keep cells at 37° C., 5% CO2 incubator for 4 hours before    harvesting.-   4. Transfer 1 mL of each condition (equivalent to 10⁶ cells) to 5 mL    FACS tube (BD) and spin at 1200 rpm for 5 min prior to fixation    (harvest the other 9 mL culture-9×10⁶ cells to 15 mL conical tube    for Western).-   5. Aspirate the supernatant and fix the cells at room temperature    with Fix/Perm Medium A (CAT #GAS001S-100) for 15 min.-   6. Wash the cells with PBS/2% FBS 1×-   7. Aspirate the supernatant and permeabilize the cells at room    temperature with Fix/Perm Medium B (CAT #GAS002S-100) for 15 min.-   8. Wash the cells with PBS/2% FBS 1×-   9. Resuspend cells with 300 uL PBS/2% BSA and divide cells into 3    tubes 100 uL each (one for isotype-Rabbit IgG Alexa Fluor-647 and    one for p-Akt Alexa Fluor-647 clone 193H12 and one for p-S6RP Alexa    Fluor-647 clone D57.2.2E, all rabbit abs are from Cell Signaling).-   10. Add 50 ng of each Ab to corresponding tube and incubate at room    temperature for 30 min.-   11. Wash the cells with PBS/2% FBS 1×-   12. Resuspend cells with 300 uL PBS/2% BSA and acquire data on    FACSCalibur (BD) using CellQuest program.-   13. Analyze data using FlowJo program and display data in    histograms.

Example 19 FACS Protocol for Measuring Quantitative Fluorescence ofApoptotic and Viable Cells Post GDC-0941 Treatment

Apoptotic and viable cells were measured by quantitative fluorescenceanalysis using fluorescence-activated cell sorting (FACS) assay withminor modifications (Munugalavadla et al (2008) Mol. Cell Biol.28(23):7182-7198). Various multiple myeloma cell lines (1×10⁶) weretreated with DMSO or various concentrations of FIG. Ia GDC-0941 for 24hr, cells were then stained using Annexin-V-APC/PI kit (BD Biosciences,San Jose, Calif.) according to the manufacturers instructions andanalyzed by flow cytometry. In case of primary multiple myeloma patientsample, 2 million nucleated BM cells were seeded in a 6 well plate in 2ml advanced-RPMI (Invitrogen, Carlsbad, Calif.) supplemented with 2%heat inactivated bovine growth serum (Hyclone, Waltham, Mass.) and 2ng/ml recombinant IL6 (R&D Systems Inc., Minneapolis, Minn.). Cells weretreated with vehicle (DMSO), 1 M or 10 M GDC-0941 for 72 hrs and thenanalyzed by flow cytometry to evaluate drug-induced apoptosis. Thefollowing reagents were used: CD45RA-FITC, CD38-APC and Propidium Iodide(all from BD Pharmingen, San Jose, Calif.). Data was acquired on aCyanADP instrument, (Dako Cytomation) and analyzed using FlowJo software(Tree Star). Live plasma cells were identified as CD38hiCD45RA- and PI-.

Example 20 Cell Cycle Analysis

Cell cycle analysis was done using Click-iT™ Edu Cytometry Assay Kit(Invitrogen, Carlsbad, Calif.) according to manufactures instructions.Fluorescence was measured on BD LSR-II and data was analyzed usingFlowJo software (Becton Dickinson).

Example 21 pAKT Measurement in Primary MM Bone Marrow Mononuclear CellsUsing Meso Scale Discovery (MSD) Assay

Bone marrow mononuclear cells (BM-MNC) from MM donors were culturedovernight in 10 ml RPMI supplemented with 10% FBS. After incubation, thecells were washed once with growth media and resuspended in 1 ml ofmedia. For each donor, the cells were split into 500 mL aliquots andwere treated with either DMSO or GDC-0941 at 1 mM for 1 hour at 37° C.After treatment, the cell pellets were collected at 1200 rpm for fiveminutes and pellets were washed once with cold PBS. The cell pelletswere resuspended with 60 ml 1× Mesoscale Discovery (MSD, Gaithersburg,Md.) lysis buffer and lysates were cleared by centrifugation at 4° C. at14000 rpm for 10 minutes. The cleared cell lysates were used to evaluatephospho-Akt (Ser473) and total Akt using MSD kit according tomanufactures instructions with minor modifications.

We claim:
 1. A method for selecting compounds to be used in combinationfor the treatment of an hematopoietic malignancy comprising: a)administering synergistic effective amounts of a therapeutic combinationof a compound selected from Formula Ia:

and a chemotherapeutic agent selected from dexamethasone, thioTEPA,doxorubicin, vincristine, rituximab, cyclophosphamide, prednisone,melphalan, lenalidomide, bortezomib, rapamycin, and cytarabine to tumorcells; b) measuring a decrease in p-Akt levels; and c) selecting asynergistic therapeutic combination for the treatment of thehematopoietic malignancy which shows a decrease in p-Akt levels.
 2. Themethod of claim 1 wherein the decrease in p-Akt level is determined byreduction of p-AKT, p-S6RP, p-Bad, or beta-actin.
 3. The method of claim1 wherein the hematopoietic malignancy is selected from non-Hodgkin'slymphoma, diffuse large hematopoietic lymphoma, follicular lymphoma,mantle cell lymphoma, chronic lymphocytic leukemia (CLL), multiplemyeloma, acute myeloid leukemia (AML), and myeloid cell leukemia (MCL).4. The method of claim 1 wherein the chemotherapeutic agent isrituximab.